Marchena overtook Brazil great Garrincha, who was unbeaten in 49 consecutive matches from 1955-66.
Marchena is not a regular starter for the European champions and came on as a substitute for Sergio Ramos in the 61st minute Saturday.
Spain has not lost a match with Marchena in the lineup since a 1-0 defeat to Greece at the 2004 European Championship in Portugal.
The Valencia defender has played 57 times for Spain.
(This version CORRECTS setting record with 50 unbeaten games.)