Biosurfactants (BSs) are mainly microbially-produced compounds that are emerging with great potentials in the formulations of biological alternatives to chemical surfactants, mainly due to their multifunctional properties, sustainability and biodegradability. Numerous investigations carried out during the past two decades have led to their involvement in many interesting potential applications including pharmaceutical and cosmetic, antimicrobial and antibiofilm, anticancer and antiadhesive active agents. BSs also have been implicated as immunomodulation and drug/gene delivery agents. Many of these potential applications are becoming essential in aspects such as the emergence of antimicrobial resistant strains or biofilm formation eradication in relation to the clinical, hygiene and therapeutic capacity and abilities to act as antibacterial, antifungal and antiviral compounds. The main thrust of research is driven by these molecules being versatile, mostly nontoxic and stable at extreme conditions. These features present BSs as potential candidates for uses in new generations of antimicrobial, antibiofilm, anticancer, wound healing, immunomodulating and drug or gene delivery agents or as adjuvants to other antibiotics and/or uses as coating molecules for surfaces as antiadhesive, suppression or eradication strategies. This chapter reviews the applications of BSs in all the above areas.