Background: Biosurfactants are amphiphilic surface active compounds that produced by several microorganisms, including, bacteria and fungi. Biodegradability, low toxicity, applications diversity and functionality under extreme conditions are characterized them from chemically biosurfactants. It is found that, Rhodotorula species, read yeasts, have high potency for biosurfactant producing. Recently, antimicrobial activities of biosurfactants have been subjected for new antibiotic therapy. The aim of the present study was to evaluate biosurfactant production by the different strains of Rhodotorula species in laboratory conditions. In addition, antifungal activity of produced biosurfactant was assessed against several saprophytic fungi. In the present study 54 strains of Rhodotorula including, R. glutinis (48 strains), R. minuta (2 strains), R. mucilaginosa (2 strains) and Rhodotorula species (2 strains) were screened for biosurfactant production. The biosurfactant was produced using the Sabouraud dextrose broth medium and confirmed by specific tests. Antifungal assay was also evaluated by disk diffusion method and the serial dilutions of biosurfactant. In the present study, although all tested strains were capable to produce biosurfactant in vitro, the degree of biosurfactant production was varied among stains. 7.4% strains had the highest (+5) biosurfactant activity followed by 16.7%, 29.5%, 25.8% and 20.4% had +4, +3, +2 and +1, respectively. In the present study, all tested fungi were inhibited at 40 µl of biosurfactant. Rhodotorula species are appropriate organisms for the production of biosurfactants and R. glutinis strains have the greatest ability to producing biosurfactant than another species. Furthermore, our results were demonstrated that the produced biosurfactant by R. glutinis presented a valuable potential for biopharmaceutical applications.