The film, which chronicles the adventures of an unwilling messiah called Brian, has been controversial since its release in 1978 when it was labelled blasphemous by religious groups.
Now the Python team are suing the film's distributor, Paragon, for allowing Life of Brian to be cut without their permission and for licensing the film at a rate that they say was not businesslike.
They are also bringing an action against Channel 4 for showing the film under licence from Paragon when, according to James Munby QC, representing Python, the station knew the team was unhappy with the deal.
In 1991 the film was licensed to Channel 4 by Python's former distributor Hand Made, owned by the Beatle's George Harrison, for pounds 250,000 with the agreement that it would be shown no more than twice over the following five years.
When the film was once more licensed to the station in 1996, by which time it had passed on to Paragon, it made just $100,000 (pounds 62,500) for a 30-year period. There were no limits to the number of times it could be shown and the deal applied to all forms of broadcasting, not just terrestrial television. Mr Munby said on behalf of Python:
"To grant rates of this sort in relation to a film for a period of 30 years when no one can foretell the nature of technology which may be in place down the line is little short of madness."
The High Court action came the day after the surviving Pythons - John Cleese, Michael Palin, Terry Jones, Eric Idle and Terry Gilliam - performed together for the first time in 18 years.
The sixth member of the group, Graham Chapman, who died 10 years ago, wrote much of the screenplay and starred as Brian, who in the film ends up on a cross while those around him sing "Always Look on the Bright Side of Life".
The Python team say that it is scenes such as this, and lines like: "He's not the messiah he's a very naughty boy", spoken by Brian's mum played by Terry Jones, which make the film ripe for censorship by those who even now fear it will give offence.
They accused Paragon of failing to include in licences an obligation that the film would not be cut.
The action, which is expected to last for three weeks, continues tomorrow.