The deal covers Cavendish Nuclear sites at Radar Road (Leicester); Aztec West (Bristol); Irlam (Warrington); Forss (Dounreay); Westlakes (West Cumbria); and Newbury (Berkshire).
It means Prospect will be able to bargain collectively for the professional, technical and some admin grades among the 500 plus staff in Cavendish Nuclear's Construction Services Group businesses.
National Secretary Gill Wood said: "This is the largest private-sector recognition deal that the union has secured since its formation in 2001 and is the result of three years of negotiations with the employer.
"It is a significant gain for Prospect and reinforces the union's collective strength not only in negotiations over members' terms and conditions but as an authoritative voice in debates over the future of the industry and UK energy policy as a whole.
"This is an exciting time for the industry as the UK stands on the edge of a nuclear renaissance, a vital component of any balanced low-carbon energy policy for the future.
"We look forward to working with all parties involved with the aim of supporting the development of high-quality, highly skilled jobs that benefit not only the communities surrounding any new plant but the overall economic growth."
Cavendish Nuclear is a wholly owned subsidiary of a global engineering services business listed on the FTSE 100 in London.