Biosurfactants are surface-active biomolecules synthesized by microorganisms. In recent years, there has been increasing interest in biosurfactants as sustainable and eco-friendly alternatives to conventional petrochemical surfactants. Due to their amphiphilic nature, biosurfactants can effectively reduce surface and interfacial tension. Biosurfactants demonstrate high biodegradability, low toxicity, and environmental compatibility. This chapter comprehensively discusses classification, physicochemical properties, and production strategies for biosurfactants, including submerged, solid-state, and co-culture fermentation systems. Special emphasis has been given to cost-effective production using renewable substrates such as agro-industrial wastes and optimization through genetic and process engineering. Among biosurfactants, the following types are most studied: rhamnolipids, sophorolipids, and surfactin. These biosurfactants have found versatile uses in such industry sectors as pharmaceutical, food, cosmetic, agriculture, petroleum recovery, and bioremediation. Their multifunctional roles include emulsification, antimicrobial activity, disruption of biofilm, and enhancement of pollutant degradation, hence making biosurfactants attract attention in circular and green biotechnological processes. In addition to many advantageous characteristics, major challenges to commercialization at large scale involve high production costs and complex downstream processing. However, recent advances in synthetic biology, improvement of strains, and valorisation of wastes open a way to large-scale commercialization. Overall, biosurfactants represent a promising class of bio-based materials with many applications that align the global drive toward sustainable industrial biotechnology and environmental stewardship.