Biosurfactants from the yeast strains Candida albicans SC5314 and Candida glabrata CBS138 were isolated and characterized. Surface tension of the cell-free broth was reduced from 72 N/m to 42 N/m and 55 N/m respectively. The biosurfactants showed emulsifying ability as the indices against castor oil were determined to be 51% and 53% for C. albicans and C. glabrata respectively and were found stable between pH 2 and 10, temperature 4–120 °C and salt concentration 2–14%. The partially purified surfactants were identified as sophorolipid using Fourier transform infrared spectroscopy. Isolated sophorolipid showed antibacterial properties against pathogenic bacteria and generated reactive oxygen species in Bacillus subtilis and Escherichia coli. Flow cytometric analysis revealed that 60 mg/L of C. glabrata biosurfactant killed 65.8% B. subtilis and 4% E. coli. The data here obtained indicates applications of biosurfactant focusing mainly as antimicrobial and therapeutic perspectives.