Introduction: Tuberculosis, caused by Methods: SPIONs were synthesized through chemical co-precipitation and functionalized with metals to create bimetallic (Ag@SPIONs, Au@SPIONs, Cu@SPIONs, Ni@SPIONs) and trimetallic (Ag-Cu@SPIONs, Ag-Ni@SPIONs) nanoparticles. The physicochemical characteristics of the metal-coated SPIONs were assessed using microscopy and spectroscopy techniques. Antimicrobial activity of the nanoparticles against Results: Transmission electron microscope analysis revealed average particle sizes of 10.28 nm for SPIONs, and 12.51 nm, 17.07 nm, 14.60 nm, 16.18 nm, 14.68 nm, and 15.00 nm for Ag@SPIONs, Au@SPIONs, Cu@SPIONs, Ni@SPIONs, Ag-Cu@SPIONs, and Ag-Ni@SPIONs, respectively, all displaying a predominantly spherical morphology. Antimicrobial testing demonstrated a potency ranking: Ag-Cu@SPIONs (1.95 µg/mL) > Ag-Ni@SPIONs (3.9 µg/mL) > Ag@SPIONs (3.9 µg/mL) > Cu@SPIONs (62.5 µg/mL) > Ni@SPIONs (>62.5 µg/mL). Gene expression analysis revealed that Ag@SPIONs and Ag-Cu@SPIONs induced strong upregulation of heavy-metal detoxification genes, whereas Cu@SPIONs significantly enriched pathways linked to valine, leucine, and isoleucine degradation, and fatty acid and propanoate metabolism. Discussion: This study demonstrates that metal-coated SPIONs possess strong antimycobacterial activity and significantly modulate key metabolic and stress-response pathways in Conclusion: Overall, the findings demonstrate that metal-coated SPIONs hold significant promise as alternative antimycobacterial agents. However, comprehensive in vivo studies are required to fully determine their therapeutic effectiveness and safety.