Ronaldo, 39, turned in a Nuno Mendes cross in the 34th minute of the match at the Estadio da Luz to reach the landmark.
He appeared emotional as he celebrated the goal, his 131st in a Portugal shirt. Half of his goals were scored for Real Madrid, with the remainder spread across his spells at Sporting Lisbon, Manchester United and current club Al-Nassr.
Diogo Dalot's goal had put Portugal in front early on before Ronaldo netted, with a Dalot own-goal reducing the deficit before the break.
The teams are in Group A1 of the latest edition of the Nations League along with Poland and Scotland.
Portugal host Scotland in Lisbon on Sunday.