Oral infections pose significant public health challenges, often exacerbating other comorbidities and increasing systemic health risks. Traditional treatments often fail to eliminate persistent micro-organisms and contribute to the rise of antimicrobial resistance. Nanoparticulate systems offer a promising solution by delivering active agents directly to targeted sites, providing more effective and localized treatment options. This study aimed to synthesize and characterize methylcellulose nanoparticles containing methylene blue at different concentrations using the nanoprecipitation method. We also evaluated their biocompatibility and antimicrobial activity against key micro-organisms commonly found in oral infections. The study involved physicochemical and morphological characterizations, including encapsulation efficiency, particle size, polydispersity index, zeta potential, and transmission electron microscopy (TEM). Additionally, controlled release profiles, antimicrobial efficacy against major oral pathogens, and biocompatibility in vitro assessments were performed. The results revealed encapsulation efficiency between 99.1 and 98.8%, with particle sizes ranging from 186 to 274 nm and a zeta potential of 1.7 to 2.9 mV achieved at lower concentrations of methylene blue and methylcellulose. The nanoparticles demonstrated sustained drug release of 85% for the smaller particles and 45% for the larger particles for more than 10 h. The nanoparticles exhibited superior antimicrobial activity compared to pure methylene blue. Cell viability studies indicated that the nanoparticles were biocompatible with approximately 40% cell viability at lower concentrations of the nanoparticles. These findings suggest that methylene blue nanoparticles could serve as a promising adjunct in dental treatments. They offer targeted antimicrobial action while potentially reducing the development of antimicrobial resistance.