<p>Structured communities of microbial cells within an extracellular polymeric matrix, called biofilms are a significant cause of the persistence and severity of chronic infection. These biofilm-mediated infections pose significant complications in the treatment plans since they are more resistant to conventional antimicrobial drugs and they are also resistant to the host immune system. Hence, new approaches should be warranted over the traditional therapies to counter such infections. The use of biosurfactants is one of the promising strategies, as these amphiphilic molecules that are produced by microorganisms are present naturally and have strong antibiofilm capabilities. Biosurfactants, including rhamnolipids, sophorolipids, and lipopeptides, work in a range of ways, including interfering with the integrity of biofilms, modulation of microbial adhesion, and quorum sensing. This review discusses the biofilm characteristics and the step of biofilm development along with the detailed analyses of the major biosurfactants and their mechanisms of action as an alternative to the conventional therapy. Moreover, we have pointed out the most recent case studies on biosurfactants with their antibiofilm activities as well as biosurfactant-coated surfaces in biofilm prevention on medical devices to provide the new opportunities in managing biofilm-related infections. Overall, this review brings the better understanding about different biosurfactants to integrate it into clinical treatments.</p>