Fare dodgers are costing Chiltern Railways £2.5 million a year, according to new figures.
The company, which released the statistic, said people who evade fares prevent it from investing more into facilities.
In a bid to clamp down on fare dodgers, Chiltern is carrying out regular checks on trains and at stations and handing out on the spot fines.
Thomas Ableman, the company’s commercial director, said they would have no hesitation taking people to court in serious cases.
Mr Ableman said: “We are committed to giving our paying customers the best service possible and take fare evasion very seriously.
“Fare evasion is an unnecessary strain on our business as it reduces funds available for passenger-focused projects and is not fair on the vast majority of our paying passengers.”
A British Transport Police spokesman added: “If you deliberately try to avoid your fare, that is tantamount to trying to steal from the rail company and your fellow passengers.
“You could find yourself being prosecuted and gaining a criminal record, which can harm your employment prospects and make it difficult to travel to some countries. Is it really worth the risk?”