The group made their first donation of £25,000 back in June 2015, just after Brighter Futures launched its appeal to raise £2.9 million to fund equipment for a new radiotherapy unit at the Great Western Hospital in Swindon.
MLOF were inspired to donate again after hearing the news last month that NHS England has now given the go-ahead for the new unit.
Chairman Robin Clark said: "The Radiotherapy Appeal is a wholly worthy cause which we are very happy to support.
"We know that there are many people living in the Malmesbury area who have to travel the 110-mile round trip to Oxford on a daily basis while undergoing radiotherapy treatment and we know how tiring and stressful it can be for them and their families.
"We want to see radiotherapy treatment brought closer to home as quickly as possible and are pleased to make this second donation to help this become a reality for local people."
Arun Desai travelled from Malmesbury to Oxford for five days a week to receive 20 radiotherapy treatments for prostate cancer.
He said: "Making the journey to Oxford would take me at least an hour each way and the effects of the radiotherapy treatment meant I had to make frequent toilet stops.
"It was a tiring and stressful experience, although the Churchill Hospital did everything they could to make the treatment times convenient for me.
"It's a wonderful idea to bring radiotherapy to Swindon and it's great that this is now becoming a reality. It will be a real bonus for a large number of people who would otherwise have to make long and painful journeys to Oxford, Salisbury or Bristol."
Jennifer Green, Head of Fundraising for Brighter Futures, said: "We are truly grateful to Malmesbury League of Friends for making a second donation to the appeal, this is a fantastic boost to our appeal and means we are now at the £400,000 mark.
"We now need more local people, businesses and organisations to get behind our appeal. Bringing cancer care closer to home will make a massive difference to so many local families.
"Contact Brighter Futures on 01793 605631 or visit our website www.brighterfuturesgwh.nhs.uk to find out how you can show your support."
The new radiotherapy unit will be an expansion of Oxford University Hospitals NHS Foundation Trust's radiotherapy service, meaning local cancer patients no longer have to make the 70-mile round trip to Oxford for treatment.
Malmesbury League of Friends provides funds to help local people with specific medical needs. The money they give to a variety of good causes comes from fundraising, donations and legacies.