George and Barbara Sutton closed the Park House Toy and Collectors' Museum in Stow-on-the-Wold in June due to ill health and a move to Berkshire.
The auction's most expensive item, a Dean's Rag Book Company "Tru-to-Life" bear from the 1950s, sold for £900.
Auctioneer Phillip Allwood, from Moore Allen and Innocent, said the collection attracted global interest from bidders.
"We had about 200 bears and not one the same - that was the one of the unusual things about this sale," added Mr Allwood.
Other lots included a Walkure bisque doll which sold for £500, a Pelham Puppet Theatre which also sold for £500 and a quantity of cake decorations which were valued at less than £80 but  sold for £480.
The museum closed in June bringing to an end the public display of thousands of individual teddy bears, dolls, puppets, train sets, model cars and tin soldiers.
"It's heart breaking to see the empty rooms, our collection was nearly 25 years in the making," said Mrs Sutton.
The couple originally travelled the country buying toys as an extension to their antiques business but the museum became the core-activity as the collection quickly grew.
Hundreds of toys which were once on display in a Gloucestershire museum have been sold at auction for £50,000.