Bale had started the defeat to Barcelona in La Liga on Saturday but the Welshman departed to whistles in the second half and was dropped for the return against Ajax, with Madrid 2-1 ahead from the first leg.
Isco came on against Barca but was not even named on the bench while Marcelo was again named as a substitute, Sergio Reguilon preferred at left-back.
Ajax started with an unchanged side from the opening leg, with Frenkie de Jong set to play at the Santiago Bernabeu for the first time. De Jong will join Barcelona in the summer.