Oyster mushrooms (Pleurotus spp.) constitute a substantial resource for functional food, as they are commercially cultivated worldwide and assumed to contain beneficial fiber. The objective of this study was to characterize and analyze the mycelia of Pleurotus ostreatus cv. Florida for its nutrient composition, total dietary fiber, insoluble dietary fiber, soluble dietary fiber content, and fermentability in vitro. Findings showed the mycelia had a moisture content of 18%, ash 12%, protein 5%, fat 0.1%, and carbohydrates 61%. Results also revealed that mycelia are an excellent source of dietary fiber (55%) containing high amounts of insoluble (22%) and soluble (33%) fiber. The mycelia also produced significant amounts of short chain fatty acids after fermentation at in vitro simulating conditions in the colon: acetate (1.92 mg/g), propionate (0.89 mg/g), and butyrate (0.31 mg/g). These results indicated that the mycelia of P. ostreatus cv. Florida are promising sources of dietary fiber.