Boris Johnson’s Deputy Mayor Richard Barnes lost Ealing & Hillingdon to Labour’s Onkar Sahota by just over 3,100 votes.
Controversial Barnet and Camden AM Brian Coleman, who also serves as Chair of the capital’s fire authority, lost to Labour’s Andrew Dismore by more than 21,000 votes.
Both men were first elected in the Assembly’s inaugural election in 2000.
Under London’s twin constituency and PR voting system the loss of the two seats meant the Conservatives retained their three ‘top-up’ list seats, securing the return of the widely admired Victoria Borwick and giving them a total of nine seats.
With opinion polls suggesting the Conservative share of the vote had fallen since the 2008 election Borwick had been expected to leave the Assembly.
Labour also gained two new list seats with Fiona Twycross and Tom Copley joining Labour’s existing Assembly Members who all retained their seats.
In total Labour now holds 12 of the 25 seats on the Assembly.
The Green Party easily retained its AMs Jenny Jones and Darren Johnson after coming third in the overall share of the list vote.
Traditionally City Hall’s third largest party, the Liberal Democrats won two seats, their lowest ever representation on the body.
Richmond councillor Stephen Knight joins Caroline Pidgeon, the only one of the three 2008-12 LibDem AMs who defended her seat, on the Assembly.