Chronic intracellular infections caused by drug-resistant pathogens pose a challenge to the treatment of chronic osteomyelitis. Such treatment requires an intracellular delivery system for the sustained release of antibiotics such as vancomycin (VCM), which is an antibiotic of last resort used against many clinically resistant bacteria. In this work, we report VCM-loaded N-trimethyl chitosan (TMC) nanoparticles and their potential application for drug delivery. The results showed that the prepared nanoparticles were predominantly spherical in shape with an average particle diameter of 220 nm, a positive zeta potential, and a loading efficiency of 73.65% ± 1.83%. Furthermore, their drug release profile followed the Higuchi model for sustained release, with non-Fickian diffusion. Over a 24-h period, 6.51% ± 0.58% of the drug within the optimized nanoparticles was released. In vitro cytology showed that osteoblasts (OBs) exhibited higher alkaline phosphatase activity (ALP) after exposure to TMC nanoparticle material. Furthermore, TMC nanoparticles increased the uptake of water-soluble quantum dots (QDs) by OBs, and both nanoparticles and VCM/TMC mixtures improved OB proliferative activity. We also investigated the minimum inhibitory concentration (MIC, 60 μg/mL), half maximal inhibitory concentration (IC50, 48.47 μg/mL), diameter of inhibition zone (DIZ, 1.050 cm), and turbidimetric (TB) assay of nanoparticles. All data demonstrated that VCM/TMC nanoparticles had excellent antibacterial activity against the Gram-positive bacterium Staphylococcus aureus. These findings suggest that VCM-loaded TMC nanoparticles have good potential for the sustained delivery of antibiotics to bone infections.