The hosts took a seventh-minute lead courtesy of a Manchester United-inspired opener, with Diogo Dalot firing past Dominik Livakovic after latching onto Bruno Fernandes's through-ball.
Portugal continued to control proceedings and grabbed a deserved second through their all-time appearance holder and record goalscorer Ronaldo.
The 39-year-old made a quick acceleration to meet Nuno Mendes's whipped delivery and guide a right-footed effort past the helpless Livakovic to register his landmark goal which brought about an emotional celebration.
After establishing complete control of the contest, Roberto Martinez's side looked set to take a two-goal cushion into the half-time break until Dalot inadvertently diverted the ball into his own net in the 40th minute.
Chelsea summer signing Pedro Neto did threaten to restore Portugal's two-goal advantage before the half-time break, but he ultimately saw his powerful shot crash off the frame of the Croatian goal.
Portugal struggled to produce the same number of chances in the second period, ensuring that Croatia held out hope of salvaging a point from their away trip.
Substitute Igor Matanovic and Andrej Kramaric both threatened in the final 15 minutes in Lisbon, but the pair both fired their respective efforts wide of Diogo Costa's goal.
Portugal ultimately stood firm to claim all three points, representing their third win in their previous four meetings with Croatia.
PORTUGAL VS. CROATIA HIGHLIGHTS
7th min: Portugal 1-0 Croatia (Diogo Dalot)
Portugal grab an early lead in Lisbon. Dalot makes a darting run into the box from the right flank to collect Bruno Fernandes's neat pass. The Manchester United defender controlled it with his first touch before slotting past Costa with his second.
34th min: Portugal 2-0 Croatia (Cristiano Ronaldo)
Ronaldo makes it two and it is a landmark goal for the Al-Nassr forward. Ronaldo guides the ball in from Mendes' inch-perfect cross to register his 900th career goal and his 131st effort for his country. The forward is visibly emotional in his celebration after creating another piece of history.
41st min: Portugal 2-1 Croatia (Dalot own goal)
Croatia pull a goal back despite being on the back foot for large periods of the first half. Dalot sticks out a foot in an attempt to deal with Borna Sosa's ball across goal, but he can only divert it past his own goalkeeper, offering Croatia a crucial foothold before the break.
MAN OF THE MATCH - CRISTIANO RONALDO
There was plenty of focus on Ronaldo's performance after failed to find the net in Portugal's Euro 2024 campaign in Germany.
At the age of 39, the veteran forward is no longer at the peak of his powers, but he demonstrated with his first-half effort that he will always possess a striker's instinct.
As well as making history with his goal, Ronaldo linked up with his teammates on a number of occasions and offered a far improved display than any of his four performances at the European Championships.
While Ronaldo may have starred for his country, the likes of Fernandes, Mendes, and Vitinha all made notable contributions to Portugal's narrow victory.
PORTUGAL VS. CROATIA MATCH STATS
Possession: Portugal 50%-50% Croatia
Shots: Portugal 15-13 Croatia
Shots on target: Portugal 6-4 Croatia
Corners: Portugal 5-7 Croatia
Fouls: Portugal 5-6 Croatia
BEST STATS
WHAT NEXT?
After kicking off their Nations League campaign with a win, Portugal will now attempt to claim a second straight victory in Sunday's home clash against Scotland.
As for Croatia, they will look to end a four-game winless run when they welcome Poland to the Opus Arena on Sunday.