In this research, a multi-step microfluidic reactor was used to fabricate chitosan – superparamagnetic iron oxide composite nanoparticles (Ch – SPIONs), where composite formation using chitosan was aimed to provide antibacterial property and nanoparticle stability for magnetic resonance imaging (MRI). Monodispersed Ch – SPIONs had an average particle size of 8.8 ± 1.2 nm with a magnetization value of 32.0 emu/g. Ch – SPIONs could be used as an MRI contrast agent by shortening T2 relaxation parameter of the surrounding environment, as measured on a 3 T MRI scanner. In addition, Ch – SPIONs with concentrations less than 1 g/L promoted bone cell (osteoblast) viability up to 7 days of culture in vitro in the presence of 0.4 T external static magnetic field. These nanoparticles were also tested against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), which are dangerous pathogens that cause infection in tissues and biomedical devices. Upon interaction of Ch – SPIONs with S. aureus and P. aeruginosa at 0.01 g/L concentration, nearly a 2-fold reduction in the number of colonies was observed for both bacteria strains at 48 h of culture. Results cumulatively showed that Ch – SPIONs were potential candidates as a cytocompatible and antibacterial agent that can be targeted to biofilm and imaged using an MRI.