Ronaldo scored from the spot in the 72nd minute but it was not enough for Portugal to stage a comeback, allowing Ukraine to pick one of two automatic tickets to EURO 2020.
Portugal are in second place but are just one point above Serbia as the race for the one remaining automatic ticket intensifies.
One consolation for Ronaldo however was his landmark strike which was his 95th goal for the Portugal national team.
The Juventus forward has scored 450 goals for Real Madrid, 118 for Manchester United, five for Sporting Lisbon, 32 for Juventus and 95 for Portugal.
"Not just anyone can reach this figure," Ronaldo told reporters.
"Thank you to my team-mates, colleagues and coaches who have helped me to become the player I am.
"How many records do I have? I don't know. I have to enjoy the moment and thank those who helped me reach this impressive mark.
The setting for Monday’s game, Kiev Olympic Stadium, was where Ronaldo won the last of his four Champions League trophies with Real Madrid in 2018 and he was disappointed not to be on the winning side this time round.
"I am disappointed that we didn't win, but proud of the team."
"The last time I played at this ground I won the Champions League, a beautiful moment that is in the past. Today we played well but couldn't win.
"Records come naturally - I don't go looking for them; records are looking for me! Without having an obsession, they happen naturally."
