Surface active agents or surfactants have a unique molecular structure, wherein a non-polar hydrophobic part, typically a straight or branched hydrocarbon chain containing 8–18 carbon atoms referred to as a tail is attached to a polar or ionic part or hydrophilic group, commonly known as a headgroup. The different interactions of these two groups in aqueous media are an important factor resulting in self-aggregation of surface-active agents into different morphological forms, viz. micelles, lyotropic liquid crystalline phases, vesicles and bilayers, etc. Surfactants sometimes are also referred as amphiphiles which in broader terms includes surface active substance like polar lipids as well. The term surfactant generally refers to man-made substance even though it contains bio-sourced molecules. Surfactants find applications in diverse aspects of chemical industries, including agrochemicals, cosmetics, detergents, dyestuffs, emulsifiers, water treatment, drug delivery systems, tertiary oil recovery, firefighting, nanotechnology, catalysis (specifically micellar catalysis) and biochemical formulations. A comprehensive account of applications of surfactants in biomedical sciences, emphasizing their advantages and disadvantages are detailed herein. Surfactants enhance solubilization of hydrophobic compounds and drugs, thereby improving bioavailability, and subsequently ensure controlled release of drug. Surfactants exhibit antimicrobial action and their antimicrobial efficacy stems from their ability to disrupt of microbial membranes. Surfactants improve stabilization of lipid nanoparticles thereby ensuring their suitability in gene and protein delivery, specifically mRNA vaccines and CRISPR-based therapeutics. Additionally, their utility in cancer diagnostics, fluorescence microscopy for biomedical imaging, enhancement of MRI contrast has attracted interest of researchers to explore their potential for theranostic applications. Surfactants facilitate scaffold fabrication and find use in biomaterial coating thereby playing a critical role in tissue engineering. However, their clinical translation is hindered by cytotoxicity, environmental concerns, and regulatory limitations.