Here, the improved efficacy of a novel composite of iron oxide (Fe3O4) magnetic nanoparticles (MNPs) functionalized by glutamic acid (Glu) and conjugated with thiosemicarbazide (TSC), evaluated on the expression of norB efflux pump gene in S. aureus strains. The proper synthesis of Fe3O4@Glu–TSC NP was confirmed and characterized by Fourier transform infrared spectroscopy (FTIR), scanning and transmission electron microscopy, Brunauer–Emmett–Teller (BET), energy dispersive X-Ray spectroscopy (EDX), X-ray diffraction (XRD), and UV–vis spectroscopy. The effect of NP on the expression of norB gene was studied using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The physical analysis of NP revealed proper synthesis, dispersion, and stability, as well as the spherical/cubic morphology with a mean size of 35–60 nm. The minimum inhibitory concentration (MIC) value of the functionalized nanoparticle decreased by two folds towards each respected strain in contrast with the Fe3O4 NP (alone). Additionally, in the presence of NP, the norB gene expression was reduced by up to 50% in comparison with the control group. In conclusion, the functionalized NP can be considered as a promising inhibitor of bacterial efflux pump genes against antibiotic-resistant bacteria. However, further studies should be conducted to determine the safety of these nanoparticles for medicinal applications.