Niamh’s Next Step chairman Chris Curry collected the donation from Daventry-based Hi-Force Hydraulic Tools' managing director last week.
Hi-Force founder Kevin Brown had been so impressed by the Wellingborough charity's presentation that he made an on-the-spot decision to donate the sum at the Northamptonshire Business Awards.
Mr Brown said: "I have got to the age of 61 years old having built my business and I want to give something back.
"We had both been nominated for awards. I was touched by the video that they showed at the ceremony. Niamh died of a rare cancer and because it's rare and it doesn't affect many people, people aren't really bothered.
"They don't just do the cancer research, they help to pay for respite care and other things that I take for granted.
"I stood up, got the microphone and said I would like to donate £25,000. My three children who are all partners in the business looked at me in shock.
"I was in quite good spirits because I had seen how good all the other nominees in our category were.
"I was so proud of my whole team when we won - from the cleaner to the head of finance we are a real people business.
"We want to be socially responsible and give back to someone who really needs it. "
Niamh's Next Step won Community Project/Person of The Year, while Hi-Force took home the Business of the Year at the awards.
Niamh's Next Step charity was founded after Chris' daughter Niamh was diagnosed with neuroblastoma - a rare cancer that affects children's nerve cells.
She died in May 2012 after an 18-month battle with the disease at the age of five years.
Mr Curry was unable to attend the evening due to illness and was surprised by friends contacting him to tell of the generous gesture.
He said: "It was just incredible that Kevin and his directors made this donation. They saw the video and wanted to donate. For someone who hadn't heard of our charity to help us is wonderful.
"The money will go toward research and children and their families living with neuroblastoma.
"We are just about to get the results of a three-year research project run by Newcastle University. They are collating the results and they look very promising.
"It's something that will 100 per cent make a difference.
"Thanks to Kevin and Hi-Force for this very generous donation."