The mayor's flagship cycle superhighway along a busy route in central London has "not been properly thought through", a business leader has said.
Howard Dawber, from Canary Wharf Group PLC, says they will create congestion for other vehicles.
BBC London has also learnt the plans include holding or "gating traffic" in outer London to reduce delays in town.
Campaigners say the scheme, which partly runs along the Embankment from Hyde Park to Tower Hill, is vital.
Cyclists make up 24% of rush hour traffic.
Andrew Gilligan, the mayor's cycling commissioner, said: "80% of journeys to and from central London are not made by car and it's about time we reshape the road network to reflect how the roads are."
But many groups have criticised how the mayor's office have carried out a consultation on the scheme, which would be the longest segregated cycleway in Europe, according to Transport for London (TfL).
The east-west route will run over 18 miles from Barking to Acton, and include a section on the Westway flyover where one lane will be removed to create a segregated cycle track.
The north-south route will run for more than three miles from King's Cross to Elephant and Castle.
Both consultations close on Sunday after being open for about two months.
Mr Gilligan said the consultation had received about 14,000 responses, of which 80% were supportive of the scheme.
The groups which have concerns include the CBI, London First, City of London and London Travelwatch.
But there are 160 companies that have signed up to the Cycling Works campaign to support them.
Steve McNamara from the Licensed Taxi Drivers Association said: "This is the last hurrah of a failed mayor. He's desperate to get this scheme in before he goes.
"This consultation is flawed and is rushed - we will certainly be looking at taking a judicial review against TfL to prevent them from going ahead with this disastrous scheme."
Mayor Boris Johnson says nothing has been set in stone.
"A great city has got to do big things for cycling but you can't be too punitive on motorists and you can't make people wait too long at either end of London.
"In the end everybody has got to get to work, everyone's got a responsibility to see their family - you've got to have compromises."