The Jubilee line is the biggest risk to transport plans for the 2012 Games, the London Assembly claimed today.
The Underground line, running from Stanmore in north-west London through to Stratford in the East End where the Games are being held, is a key route to the Olympic Park and other Games venues.
The line has been marred by heavily delayed upgrade works and regular disruptions, the London Assembly Transport Committee said.
Infrastructure failures, which can include delays caused by automatic train operating systems, fleet, signals, track and stations, increased by 12% between March and December last year compared with the same period in 2010.
The committee points out that by contrast all the other London Underground lines recorded a drop in delays due to infrastructure failures over the same period.
Extra trains will be introduced on March 25 but the committee said it was concerned about whether this leaves enough time for testing.
The committee has now written to the London mayor to ask for details of how officials will ensure the line will be up to scratch in time for the Games.
There are also fears the Olympic Route Network (ORN) could confuse motorists and worsen traffic congestion.
Transport for London (TfL) is now in the final stages of setting up the 109-mile (175km) ORN, which will include 30 miles (48km) of expressway Games lanes.
The ORN is set to open from July 25, two days before the start of the Games, from 6am to midnight.
It will be opened up to all traffic at times when demand from Games family vehicles is low and capacity available. A network of 150 variable message signs along the route will tell motorists when the Games lanes are open to regular traffic throughout the day.
Committee chairman Caroline Pidgeon said: "Given the scale of the challenges, some disruption to the transport network is inevitable.
"Keeping the capital moving smoothly during the Games will be nothing short of a Herculean task.
"The amount of work that is going in to getting our already overburdened transport network ready to carry millions of extra athletes, officials and spectators is very impressive.
"We are, however, keen to get some reassurance about the few areas - like the Jubilee line, local parking arrangements and the Olympic Route Network - that are still letting down what are otherwise very robust preparations."
Mark Evers, TfL's director of Games transport, said: "We welcome the Assembly's assurance that we have made good progress on preparing the transport network ahead of this summer.
"We know that hosting the London 2012 Games will be a huge logistical challenge, but we have a detailed plan and we are confident that we will get everyone to their Games events on time and keep London and the UK moving.
"On the Tube, delays have been cut by around 40% since 2007/08, with the Tube now running more reliably and carrying more people than ever before.
"Enormous strides have been taken to improve the reliability of the Jubilee line following the completion of works to upgrade the line's signalling system.
"Delays - as measured by the number of passenger hours lost to disruption - have more than halved compared with the same period last year.
"Further targeted work is being undertaken on the line to continue to improve reliability."