Surfactant-based formulations have applications from biological fields to industry. Chemical surfactants are often harsh and have high toxicity, low biocompatibility, and poor biodegradability. As a result, they cause significant environmental problems and ecological damage. Therefore, eco-friendly biosurfactants, mainly derived from microorganisms, are preferred as they help maintain environmental safety. Biosurfactants require lower concentrations to reduce surface and interfacial tension, indicating a lower CMC than commonly used surfactants. Biosurfactants remain stable in various salt concentrations, different pH levels, and temperatures, and they stabilize emulsions more effectively than chemical surfactants. The chapter also covers the production of biosurfactants, including microbial strains, fermentation strategies, and factors affecting yield. Biological activities of biosurfactants, including antimicrobial and antifungal effects, indicate their potential applications in medicine, agriculture, and the pharmaceutical industry. Overall, biosurfactants offer significant industrial and environmental advantages, and future research may focus on large-scale production, economic feasibility, and the development of novel applications.