Council leader Phelim Mac Caffety and deputy leader Hannah Allbrooke have lost their seats amid a Labour landslide across the council.
The pair had served as councillors in Brunswick and Adelaide, but have been ousted by Labour candidates Andrei Czolak and Jilly Stevens.
Ms Allbrooke lost by just six votes in the ward, while Mr Mac Cafferty slumped to just 901 votes.
Ms Allbrooke sprinted off the stage immediately after the result was announced, followed shortly after by Mr Mac Cafferty.
Speaking after the result, newly-elected Councillor Czolak said: “If you told me this result at the start of the campaign, I wouldn’t have believed you.
“The odds were stacked against us - Labour had not won in Brunswick since 1996.”
He said support on the doorstep had been “really positive” and said: “It was tough to get through, but I am just so happy.”
The result in Brunswick and Adelaide had been subject to a recount due to the close race between Ms Allbrooke and Cllr Czolak.
Labour now have 37 councillors on Brighton and Hove City Council, with the Conservatives on six and the Greens on five.
The Brighton and Hove Independents have two councillors, with one independent elected - Peter Atkinson in North Portslade.
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