The capacity to tune the degree of mucoadhesion and mucopenetration of nanoparticles is essential to improving drug bioavailability, transport, and efficacy at mucosal interfaces. Herein, self-assembled nanoparticles (NPs) fabricated from amphiphilic block copolymers of poly(lactic acid) (PLA) and poly(oligo(ethylene glycol) methacrylate) (POEGMA) with various side chain lengths (PLA-POEGMAn) are reported to facilitate tunable mucosal interactions. PLA-POEGMAn nanoparticles with long PEG side chain lengths (n = 20, or 40) demonstrated mucoadhesive properties based on rheological synergism, calorimetric tracking of mucin-nanoparticle interactions, and the formation of larger NP-mucin hybrid structures; in contrast, NPs fabricated from block copolymers with shorter PEG side chains (n = 2/8-9 or n = 8,9) showed poor mucoadhesion but penetrated through the mucin layer with significantly higher permeation rates (>80%). All NP formulations showed good cytocompatibility (viability > 70%) with human corneal epithelial cells in vitro and no detectable acute in vivo ocular irritation in Sprague-Dawley rats. Coupled with the capacity of the synthetic route to easily incorporate different brush lengths and/or different functional groups into the hydrophilic block, we anticipate this approach may offer a solution in applications in which balancing mucoadhesion and mucopenetration is critical for enabling effective drug delivery.