Background: Osteoarthritis (OA), affecting approximately 240 million people worldwide, currently lacks targeted, long-acting therapeutic options. This study investigates how physicochemical properties (i.e., size, surface charge, polydispersity) influence poly(lactic-co-glycolic acid) (PLGA) microsphere (MS) diffusion into articular cartilage for enhanced corticosteroid delivery. Methods: PLGA MSs were synthesized via oil/water emulsions to create a variety of sizes and surface charges. MSs were loaded with corticosteroid (H-17-B) and release kinetics were studied in vitro and analyzed via HPLC. Diffusion of the MSs was investigated via a bovine explant model. Results: Unmodified PLGA MSs (approximately 0.58–0.98 µm) were synthesized via single-stage oil/water emulsion with varying poly(vinyl alcohol) concentrations and sonication intensities. All formulations exhibited near-neutral surface charges (−0.27 to 4.28 mV). Smaller particles achieved greater cartilage penetration, with bi-exponential diffusion models (R² = 0.706–0.999) outperforming classical Fickian approaches. However, multi-timepoint validation demonstrated fundamentally non-diffusive transport, likely governed by steric exclusion from the dense collagen network (0.05–0.06 µm pore size). Surface functionalization with avidin/palmitic acid or polyethylene glycol (PEG)/biotin yielded microspheres with controlled properties (0.36–0.97 µm; PDI: 0.10–0.32). In vitro release studies with hydrocortisone-17-butyrate (H-17-B; encapsulation efficiency of 79.8% ± 4.8%) demonstrated biphasic kinetics best fit by bi-exponential models (R² > 0.95). Unmodified microspheres exhibited 7.1% cumulative release by Day 14. High-performance liquid chromatography revealed that H-17-B undergoes ester hydrolysis to hydrocortisone during release, with surface modifications significantly affecting drug stability. Specifically, PEGylated microspheres maintained 96% of the drug in H-17-B form at Day 14 compared to only 37% for unmodified particles. Release was governed by PLGA degradation with concentration-independent kinetics, enabling predictable dose scalability. Conclusions: This work establishes that the behavior of PLGA microspheres in cartilage is controlled by size, charge, and surface functionalization. Surface modifications overcome physical barriers while stabilizing the encapsulated corticosteroid against premature hydrolysis, providing a framework for designing intra-articular drug delivery systems for osteoarthritis treatment.