Background: Biosurfactants are natural surface-active compounds produced by a variety of microorganisms. The high cost of culture media limits the large-scale production and use of biosurfactants. It is therefore necessary to develop an efficient and cost-effective bioprocess to improve the yield of biosurfactants from microorganisms. In this study, the response surface method was used to optimize the production of biosurfactants by a Lactobacillus strain and the antimicrobial activity of the biosurfactants was assessed. Results: The biosurfactant-producing strain was identified as Lactobacillus paracasei subsp. tolerans N2 after 16S rRNA gene analysis. Among the different variables studied using a Plackett-Burman statistical design, temperature and peptone and sugar cane molasses concentrations were found to be the main factors that had significant (p < 0.05) influence on biosurfactant production. The results of this study showed that molasses concentration at 59.5 g/L, peptone at 6.20 g/L and temperature of 33 °C were optimal conditions for biosurfactant production, with a maximum yield of 2.70 g/L. The biosurfactant exhibited surface tension reduction of 37.85 mN/m and antimicrobial activity expressed as inhibition diameter of 63 mm. Partial characterizations by elemental, biochemical and Fourier transmission infrared spectroscopy analysis of the biosurfactant produced revealed that it was glycolipoprotein in nature. The biosurfactant exhibited bactericidal activity against Pseudomonas aeruginosa PSB2, Pseudomonas putida PSJ1, Salmonella sp. SL2, Escherichia coli MTCC 118, Bacillus sp. BC1 and Staphylococcus aureus STP1 at concentrations ranging from 6.4 to 50 mg/mL. Conclusion: The yield of biosurfactant was four-fold higher after optimization of media components and culture conditions using response surface methodology. The results of this study suggested that sugar cane molasses can be used as a low-cost substrate to enhance the yield of biosurfactants with antimicrobial activity.