The pair are the first new arrivals at the site since construction work to expand the neighbouring sewage works began in spring 2010.
Experts have ringed the owls and will monitor their development.
Karen Sutton, manager of Thames Water’s Crossness Nature Reserve, said: “The last time we had barn owls breed at Crossness was in 2007 so we were over the moon to see the arrival of Plop and Flo, in the same box our owls have bred in the past.
“Fortunately the major upgrade work under way here has not put off our breeding barn owls. We put a number of new boxes up around the site to encourage them to set up home. Not only have the barn owls made themselves at home in the lower section of one box, but a family of kestrels have also set up home upstairs.”
Barn owls are protected by law and their numbers have declined drastically in recent years as a result of rodent poisons and the loss of traditional nesting and roosting sites.
Thames say barn owls were first spotted at Crossness, which is one of Europe’s largest sewage works, in 2005 when they bred on the site for the first time.
The company says the facility’s upgrade, scheduled for completion in 2014, will boost treatment capacity by 44 per cent and “significantly” reduce the amount sewage which overflows into the Thames.
Thames has also planted new reed beds at the works to house a family of water voles currently being looked after at a captive breeding centre in Kent.