"AS Roma can confirm head coach Eusebio Di Francesco has left the club with immediate effect", the club said in a tweet.
Tiquinho Soares put Porto ahead in Portugal before Daniele De Rossi levelled from the penalty spot, but Marega's 52nd-minute goal ultimately forced extra time.
Di Francesco, 49, was part of the last title-winning Roma team as a player in 2001 and took over the club in the summer of 2017.
A 117th minute penalty goal from Alex Nicolao Telles handed FC Porto a 3-1 win over AS Roma in a UEFA Champions League Last 16, second leg match at the Estadio do Dragao on Wednesday.More news: Tesla Says New Supercharger Cuts Charging Time By 50%
"On behalf of myself and everyone at AS Roma, I'd like to thank Eusebio for his work and his commitment. We all wish him well for the future".
Domestically, Roma now sit fifth in the Serie A table, three points behind Inter for the final Champions League place.
Shortly afterwards, Roma also thought they had won a penalty when the referee stopped the game while the video assistant looked for a possible push by Moussa Marega on Patrik Schick but play was waved on, this time without a pitchside review.
Blues owner Roman Abramovich is set on Champions League qualification next season and that continues to remain in the balance.
"Last year we asked for VAR in the Champions League because we got screwed in the semi-final and tonight, they've got VAR and we still get robbed", Pallotta posted on the club's official Twitter feed. I'm exhausted of this c**p.