Gian Piero Gasperini's side finished second in Group D to book their spot in the knockout stages, while Real Madrid ultimately topped Group B after a shaky start to proceedings.
Had this fixture been played five years ago, Real Madrid would have been expected to sweep aside their Italian counterparts on their way to yet another European crown. However, Atalanta's meteoric rise has their fanbase believing that a shot at continental glory is certainly on the cards.
La Dea's maiden Champions League campaign last season ended with quarter-final heartbreak at the hands of beaten finalists Paris Saint-Germain, but Gasperini's free-scoring charges have not let up this term and managed to qualify in second place from Group D - behind Liverpool, whom they memorably beat at Anfield - and above 2018-19 semi-finalists Ajax.
Atalanta have been rewarded for their efforts with one of the biggest ties in the club's illustrious 113-year history, and the Bergamo-based outfit will welcome an injury-hit Real Madrid to Italy with genuine belief that they can get one over the most successful team in Champions League history.
As far as their domestic fortunes are concerned, Gasperini's side are some way off the two title-chasing Milan clubs in Serie A, but they warmed up for this game with a thrilling 4-2 win over Napoli, with Colombian compatriots Duvan Zapata and Luis Muriel both adding to their already impressive tallies for the season in that one.
The former starred in La Dea's group stage ventures with three goals and two assists from his side's six outings, and Atalanta have notched up at least three goals in nine of their last 11 home outings, so a ravaged Real Madrid defence could be in for a taxing evening at the Gewiss Stadium.
Real Madrid's perennial reign of dominance in the Champions League over the past decade quickly became a thing of the past when they lost to Shakhtar Donetsk in their inaugural fixture of the tournament, and it was anyone's game in Group B heading into the final matchday.
However, Zinedine Zidane's men displayed their credentials with a comfortable 2-0 win over Borussia Monchengladbach to propel themselves to top spot in a group where Inter Milan finished rock bottom, but the 13-time European champions are certainly vulnerable this season.
Since their most recent Champions League triumph in the 2017-18 season, Real Madrid have been knocked out at this stage to Ajax and Manchester City respectively, and Zidane's decimated team cannot afford to let a hint of complacency creep in if they are to travel home from Bergamo with a first-leg advantage.
The La Liga champions have managed to win their last four on the bounce domestically - keeping clean sheets in their last three outings - and their winner against Real Valladolid at the weekend came from an unlikely source in Casemiro, who ensured that Real would take of advantage of Atletico Madrid's defeat to Levante and narrow the gap at the top to three points.
Wednesday's battle represents the first ever meeting between Atalanta and Real Madrid, and even though the visitors boast 13 Champions League titles compared to La Dea's empty European cabinet, only a brave soul could back Zidane's side to comfortably prevail in this fixture.
Atalanta BC Champions League form: WDLWDW
Atalanta BC form (all competitions): LDDWWW
Real Madrid Champions League form: LDWWLW
Real Madrid form (all competitions): WLWWWW
Atalanta's only confirmed absentee through injury at the minute is right-wing back Hans Hateboer, and his deputy Joakim Maehle shrugged off previous fitness concerns to play 90 minutes against Napoli.
Two of Gasperini's back three in Cristian Romero and Berat Djimsiti are walking a tightrope with two bookings apiece, so Jose Luis Palomino is ready to be called upon if the hosting manager does not risk either player being banned for the second leg.
La Dea have a wealth of talent in their midfield ranks and Gasperini has a big call to make when it comes to who will play behind Zapata and Muriel, but Josip Ilicic could get the nod ahead of Matteo Pessina.
In stark contrast, Real Madrid's infirmary is at full capacity right now, and the visitors were dealt a further blow ahead of this match with Karim Benzema - the Champions League's fifth-highest goalscorer - confirmed to be missing through injury.
The Frenchman joins the likes of Eden Hazard, Rodrygo, Sergio Ramos, Marcelo, Eder Militao, Daniel Carvajal and Federico Valverde in the treatment room, while Casemiro is one yellow card away from a suspension.
With only 12 recognised senior players in the squad for the trip to Italy, Zidane's XI picks itself for the first leg.
Atalanta BC possible starting lineup:
Gollini; Toloi, Djimsiti, Palomino; Maehle, De Roon, Freuler, Gosens; Ilicic; Zapata, Muriel
Real Madrid possible starting lineup:
Courtois; Vazquez, Varane, Nacho, Mendy; Casemiro, Kroos, Modric; Asensio, Diaz, Vinicius Jr
We say: Atalanta BC 3-1 Real Madrid
A full-strength Real Madrid would be capable of making the first leg a truly tantalising battle, but Los Blancos' decimated defence will no doubt struggle to contain Atalanta's rampant attacking contingent. The hosts' rearguard is not impenetrable by any means, but Gasperini's men will smell blood and we expect them to march to a historic triumph over their opponents before they travel to Madrid for the second leg next month.