Abstract Background Linezolid (LNZ) is extremely prone to resistance. The development of resistance to LNZ should be taken into consideration when selecting this drug as a therapeutic option. It is well established that reactive oxygen species (ROS) generated by iron oxide nanoparticles (MNPs) could kill the infecting bacteria. So, we hypothesized the synergistic antibacterial effect of iron oxide nanoparticles and LNZ. Objective To study the release and antibacterial effects of LNZ-loaded superparamagnetic iron oxide nanoparticles (SPIONs) on Staphylococcus aureus and Streptococcus pneumoniae. Method Ferrofluid containing SPIONs was synthesized via chemical co-precipitation method and stabilized by sodium lauryl sulphate (SLS). SPIONs were then loaded with LNZ and characterized for particle size, FT-IR, XRD, and entrapment efficiency. Further antibacterial activity of SPIONs and LNZ-loaded SPIONs was investigated. For the in vitro release findings, HPLC analytical method development and validation were performed. Results Isolation of LNZ was accomplished on a C-18 column with methanol–TBHS (tetra butyl ammonium hydrogen sulphate, 50:50, v/v). The eluate was monitored at 247 nm with a retention time of 4.175 min. The MNP’s DLS measurement revealed monodispersed particles with an average size of 16.81 ± 1.07 nm and PDI 0.176 ± 0.012. In optimized formulation, 25 ± 1.75% (w/w) of the drug was found to be entrapped. XRD revealed uniform coating of oleic acid covering the entire magnetic particles’ surface with no change in its crystallinity. An effective antimicrobial activity was observed at the lowered dose of drug. Conclusions A robust HPLC method was developed to quantify the LNZ in MNPs, and outcomes showed that the reduced dose of LNZ incorporated in SPIONs was able to show similar activity as the marketed product. Highlights Successfully reduction of the dose of LNZ was established with the aid of biocompatible MNPs to attain the equivalent antibacterial activity.