Background: Biosurfactants are amphiphilic surface-active compounds that are produced by several microorganisms, including bacteria and fungi. Biodegradability, low toxicity, application diversity, and functionality under extreme conditions characterize them from chemical biosurfactants. It is found that Rhodotorula species, read yeasts, have high potency for biosurfactant production. Recently, antimicrobial activities of biosurfactants have been subjected to new antibiotic therapy. Objectives: The aim of the present study was to evaluate the biosurfactant production by the different strains of Rhodotorula species in laboratory conditions. In addition, the antifungal activity of produced biosurfactant was assessed against several saprophytic fungi. Methods: In the present study, 54 strains of Rhodotorula including R. glutinis (48 strains), R. minuta (two strains), R. mucilaginosa (two strains), and Rhodotorula species (two strains) were screened for biosurfactant production. The biosurfactant was produced in Sabouraud dextrose broth medium and confirmed by specific tests. The antifungal assay was carried out by a disk diffusion method using serial dilutions of biosurfactant. Results: In the present study, although all tested strains were capable of producing biosurfactant in vitro, the degree of biosurfactant production varied among the strains. 7.4% of the strains had the highest (+5) biosurfactant activity while 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 the biosurfactant. Conclusions: Rhodotorula species are appropriate organisms for the production of biosurfactants and R. glutinis strains have the greatest ability to produce biosurfactant among other species. Furthermore, our results demonstrated that the produced biosurfactant by R. glutinis presents a valuable potential for biopharmaceutical applications.