<p>Biofilm-related infections represent a long-standing problem in clinical and industrial environments because of their innate resistance to standard antimicrobials. Biosurfactants, which are amphiphilic and biodegradable compounds made by microorganisms, are showing great promise as anti-biofilm agents due to their wide-ranging antimicrobial effects and minimal toxicity. They function by reducing bacterial adhesion, destabilizing the structural integrity of the extracellular polymeric substance matrix, and enhancing membrane permeability, which may ultimately lead to cell lysis. The multifunctional characteristics of biosurfactants facilitate a wide range of biomedical applications such as medical device coatings, drug delivery systems, synergistic antimicrobial formulations, and vaccine adjuvants. Their compatibility with the environment makes them promising alternatives to synthetic surfactants. However, limitations such as low production yields, expensive purification, and regulatory uncertainties are deterring large-scale commercial usage at present. Future studies need to be directed towards metabolic and genetic engineering approaches to increase the yield of biosurfactants, utilize omics tools to define their specific mechanisms, and use nanotechnology-based drug delivery systems for enhanced biofilm disruption. Biosurfactants thus represent a sustainable and innovative solution against biofilm-associated infections and antimicrobial resistance.</p>