Two first-half goals from Evan Ferguson put the Seagulls in control at the Amex Stadium, before the already-relegated Saints pulled one back through Mohamed Elyounoussi just before the hour mark.
Theo Walcott thought he had restored parity, only to ruled offside by VAR, before Pascal Gross netted in the 69th minute to help Brighton guarantee a top-seven finish and qualify for Europe for the first time in their history.
In front of a jubilant Amex Stadium crowd, De Zerbi was handed a microphone after the match to thank the club's supporters for their efforts throughout this season and insisted that his side still have one final target heading into the final two games of the season.
"You have been fantastic, one player more. We need another point to play Europa League because we want to play in the Europa League, not the Conference. See you Wednesday," said De Zerbi.
Speaking to BBC Sport after the match, De Zerbi described Sunday's victory as 'one of the best days in his career' after leading Brighton into Europe and to their highest-ever top-flight finish.
"It's incredible, one of the best days in my career. I can change my ideas and philosophy depending on the result but I think we deserve to qualify for Europe," said the Italian.
"We need one more point to qualify for the Europa League and we have to do it, but I have a lot of emotion. I am really proud to be coach of these players and the club. We won together, and I am really proud.
"I can't explain my emotion, my players are fantastic players, but also fantastic people. They believed from the first moment that they deserved to qualify, we had a tough period, we lost a lot of players to in jury but when you have something inside you push to the end top your target.
"I did not have time to think about a target, I only focused on my improvement and my work. After the game with Palace away, I understood this could be the right season and the right year to achieve one incredible, historic target.
"We did not win and the players were sad, but that was when I understood their level because only with the level of these players can you achieve this historic target."
Meanwhile, Gross has reflected on 'the best day of his football life' after become Brighton's joint-record scorer in the Premier League, drawing level with Glenn Murray on 26 goals.
Speaking to the Seagulls supporters, as quoted by The Argus, Gross said: "It's a dream come true for all of us, the whole club. Especially for the team, the supporters, everyone who works at the club. It's the best day of my football life."
Brighton will play host to Premier League champions Manchester City in their final home game of the season on Wednesday night, before travelling to Villa Park to face Aston Villa on the final day next weekend.