Barley protein (BP) was extracted from barley flour, and a BP film was prepared. To improve the physical properties of the BP film, nano-clay was incorporated. Among the films prepared, the composite film containing 4% BP/1% Cloisite Na+ had the best physical property. The composite film containing grapefruit seed extract (GSE) was prepared and used as a packaging film for the button mushroom. Packing of the mushroom with this film inhibited microbial growth during storage. After 7 days of storage, packaging of mushrooms with the film containing 0.7% GSE reduced the populations of total aerobic bacteria and yeast and moulds by 0.95 and 0.58 log CFU/g, respectively, compared with the control. These results suggest that the BP/Cloisite Na+ film containing GSE can be used as an environment-friendly packaging for maintaining mushroom quality during storage.