Senad Lulic tapped home the winning goal from close range in the 70th minute after Roma goalkeeper Bogdan Lobont tipped Antonio Candreva's cross into his path.
The victory means Lazio will qualify for the Europa League, salvaging an otherwise disappointing season and rubbing further salt into the wounds of their old foes, who miss out on European football despite finishing sixth one place clear of the Biancocelesti.
"I am really happy for the players who worked so hard this year and deserved the victory. It's not easy to win a game like this. We now have Europe ahead of us," Lazio manager Vladimir Petkovic told RAI Sport.
The defeat for Roma makes their manager Andrea Andreazzoli's position at the club precarious with speculation in the lead up to the match suggesting AC Milan coach Massimiliano Allegri could be a favourite to take his place.
"I don't think it's the time to be talking about my future," Andreazzoli told the post-match news conference. "It was a bad match to be honest. Neither side created much and there were a lot of errors.
"In the second half when it seemed we were in control Lazio punished us with the once chance that often decides these matches."
Roma attacker Mattia Destro, who was otherwise isolated up front, nearly struck in first half stoppage time when he stretched to meet Marquinho's cross and sent his header wide of the target.
Roma thought they had equalised when captain Francesco Totti's free-kick was deflected onto the bar, only for Lazio stopper Federico Marchetti to snatch the ball away from a manic goalmouth scramble.
Lazio contained Roma's late pressure with relative ease as they desperately searched for an equaliser and held out to record a famous victory.
