The Dutch giants lost their last outing and are one point behind Atalanta, meaning that only a victory will do on Wednesday night if they are to leapfrog their opponents into second place.
Ajax's defeat to Liverpool last week, combined with Atalanta's draw with Midtjylland, means that it is all to play for in Group D.
In what could prove to be a blessing or a curse, Atalanta had the weekend off as their game against Udinese was called off due to a waterlogged pitch.
Ajax were in Eredivisie action on Saturday and saw their impressive run of league form come to an end in a 2-1 home loss against Twente.
Erik ten Hag's charges now need to pick themselves up for this big showdown as they look to reach the knockout stages for the first time since 2018-19 when making it to the semis.
Atalanta made it to the last eight in the most recent edition of the competition and boast an incredible away record in Europe, winning four in a row on their travels in the Champions League.
However, Ajax are unbeaten in their last seven games against Italian opponents in UEFA's flagship competition, including a 2-2 draw with Atalanta in the reverse fixture on matchday two.
Ajax Champions League form: LDWWL
Ajax form (all competitions): WWWWLL
Atalanta BC Champions League form:
Atalanta BC form (all competitions): LDDWLD
Sean Klaiber, used from the bench three times in this year's competition, is in contention for a start at right-back in the absence of Mazraoui.
Playmaker Dusan Tadic has been involved in five of Ajax's seven Champions League goals this season, scoring two and assisting three.
Ajax possible starting lineup:
Onana; Klaiber, Schuurs, Blind, Tagliafico; Klaassen, Alvarez, Gravenberch; Antony, Traore, Tadic
Atalanta BC possible starting lineup:
Sportiello; Toloi, Romero, Djimsiti; Hateboer, De Roon, Freuler, Mojica; Gomez; Zapata, Muriel
We say: Ajax 1-1 Atalanta BC
Five of Ajax's last seven games against Italian sides have ended all square.
That result would not be enough to see them through, but Atalanta have been impressive on their travels - even winning at Anfield last time out - and we can see the Serie A side picking up the point they need.