A coffin was carried by London's businessmen and women to City Hall in protest at the congestion charge rise.
The mayor said up to £45m will be raised with the increase
A petition signed by more than 5,000 residents was also handed into the mayor of London's offices during Wednesday morning's protest.
Protesters claim the increase from £5 to £8 will be "the death of London's small businesses". The mayor said it would cut traffic by a further 5%.
A campaign spokesman said the move was "simply a money-raising exercise".
Cut trafficThe increase will come into force on 4 July. Charges for fleet vehicles will rise to £7 and there will be discounts for people who pay monthly or annually.
The mayor said up to £45m would be raised which will help cut traffic levels and improve public transport.
Gordon Taylor, who owns an engineering company in Chelsea, said the mayor was taking "millions out of the private sector and putting it into Capita (which administers the congestion charge) and local transport.
"This money has to come from somewhere and it's the local businesses which will suffer.
"We rely on deliveries to and from our business which will cost us more with the increase."
Up to 30 businessmen and women will took part in the protest.