Mucosal drug delivery offers a promising non-invasive approach in pharmaceutical sciences but faces challenges from the protective mucus layer on mucosal surfaces, limiting drug absorption. Polymeric nanoparticles (NPs) have emerged as effective carriers, leveraging their small size to enhance mucus penetration and drug absorption. This study investigates polyvinyl alcohol (PVA)'s role in enhancing poly(lactic-co-glycolic acid) (PLGA) NPs for mucosal drug delivery. While PVA is commonly used as an emulsion stabilizer, its impact on NP mucoadhesiveness and mucodiffusivity is often overlooked. We characterized PLGA-based NPs with varying PVA types, employing particle tracking and molecular dynamics to demonstrate PVA's direct interaction with mucin MUC 5B. Our findings show reduced interaction with lower PVA hydrolysis, with particle tracking confirming PVA properties influence mucoadhesion. This study underscores PVA's critical role in optimizing NP efficacy for mucosal drug delivery, offering insights for developing more effective drug delivery systems.