The Real Madrid forward, one of the brightest players on the pitch before he departed the action at half time, struck on 36 minutes to settle the match.
The deadlock at the Stade de Genève was broken as the Portugal captain fired home from Silvestre Varela's lay off and he went close to adding a second soon after when a downward header bounced up and came back off the cross bar.
Ronaldo's withdrawal was one of nine changes seen in the second half as both Portugal coach Paulo Bento and his Croatian counterpart Igor Stimac took the opportunity to look at new options.
One of them was 16-year-old Dinamo Zagreb midfielder Alen Halilovic, who made his international debut from the bench and nearly snatched a draw for Croatia as time ran out, forcing a smart save out of Portugal keeper Eduardo.
Portugal held on for the win - their second 1-0 success on the bounce following Friday's World Cup qualifying win over Russia - and will return their focus to qualification from Group F against Northern Ireland in September.
Defeat for Croatia was their second reverse in as many games after they were stunned by Scotland in their Group A qualifier on Friday. Next up for Stimac's side, who lie second in the table, are Serbia in September.