At present, the universal health problem with Staphylococcus aureus is the emergence of multidrug-resistant strains due to the overuse of antibiotics. Drug extrusion through efflux pumps is one of the bacterial mechanisms to neutralize the bactericidal effect of antibiotics. The antibacterial activity of silver nanoparticle as well as Fe3O4 nanoparticle had been previously studied and widely described. Today, the development of green methods for nanomaterial synthesis is an important aspect of research in the field of nanotechnology. Here, we report the biosynthesis and characterization of Fe3O4@Ag nanocomposite by Spirulina platensis cyanobacterium and it impacts on the expression of efflux pump genes in ciprofloxacin-resistant S. aureus (CRSA). The physical properties of biosynthesized nanocomposite measured and confirmed by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and scanning and transmission electron microscopy. The minimum inhibitory concentration (MIC) of ciprofloxacin in CRSA strains was determined in the presence of Fe3O4@Ag nanoparticles by broth microdilution method. The effect of Fe3O4@Ag nanocomposite on the expression of norA and norB genes was evaluated by real-time PCR. The physical analysis confirmed well-dispersed, highly stable, and mostly spherical Fe3O4/Ag NPs with the average size of 30–68 nm. The results of antibacterial tests showed the synergistic effects of nanocomposite and antibiotics in MIC reduction. Additionally, in the presence of Fe3O4@Ag nanocomposite, the expression of norA and norB genes was decreased more than twofold compared to control. In conclusion, the Fe3O4/Ag nanocomposite can use as an effective inhibitor of antibiotic resistance in medicine.