Biosurfactants are amphiphilic molecules with unique surface-active properties that have emerged as promising agents for drug absorption by enhancing drug solubility, permeability, and stability in biological environments. Specifically, they address challenges in delivering Biopharmaceutics Classification System (BCS) Class II/IV drugs through mechanisms such as micellar solubilization (rhamnolipid-based encapsulation), permeability enhancement, cellular uptake facilitation, bioadhesion, and drug protection from enzymatic degradation. These properties enable targeted drug delivery (biosurfactant-coated nanoparticles), controlled release, and reduced systemic toxicity, making biosurfactants suitable for application in cancer therapy, antimicrobial treatments, nanomedicine, and gene therapy. However, several challenges, including high production costs, stability issues, limited mechanistic understanding, and regulatory hurdles, restrict their widespread use in pharmaceuticals. Advancements in genetic engineering, fermentation technology, and responsive formulations (pH-sensitive liposomes) can overcome these limitations. This chapter explores the mechanistic aspect of drug absorption for poorly soluble drugs in association with biosurfactants. Future research must focus on optimizing biosurfactant functionality, ensuring safety through rigorous clinical validation, and harmonizing regulatory frameworks to unlock their full potential as sustainable alternatives to synthetic surfactants.