Antibiotic resistance is a growing concern due to the overuse of antibiotics. Alternative treatments, such as nanoparticles, are being explored. Biological synthesis of iron oxide nanoparticles (Fe3O4-NPs) via probiotics offers a sustainable and cost-effective method over the toxic chemical approaches, but there are challenges regarding its heavy metal resistance and the toxicity of the obtained nanoparticles. Thus, this research aims to biologically synthesize Fe₃O₄-NPs via a new bacterial isolate and evaluate its toxicity. The objectives are to isolate and characterize a novel bacterial isolate with probiotic potential. Then, to biologically synthesize and characterize Fe3O4-NPs via Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FE-SEM), Powder Diffraction Techniques (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), and nanosizer. Finally, to evaluate its cytotoxicity potential via MTT– (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. A total of 12 samples were collected from six different sites in Selangor, Malaysia. The 16s rRNA sequencing came closest to Bacillus proteolyticus UPMC1508 (99.87%). This strain has lower antibiotic resistance and high Fe-tolerance (MTC = 0.8 mg.mL− 1). It successfully synthesized Fe3O4-NPs, which exhibited absorption curves between 290–300 nm. TEM and FE-SEM indicated spherical formed Fe3O4-NPs; the average diameter was 5.12 ± 0.95 nm. Meanwhile, XRD peaks revealed that the grain size was around 32.61 nm. The nanosizer revealed a hydrodynamic diameter of around 104 nm with a good Polydispersity index (PDI) value (0.217). FT-IR indicated a satisfactory stability of Fe3O4-NPs after 2 months. Finally, Fe3O4-NPs showed low toxicity at 0.031 mg.mL− 1.The findings revealed that the novel isolated B. proteolyticus UPMC1508 has high Fe-heavy metal tolerance and less antibiotic resistance. Furthermore, it successfully synthesized Fe3O4-NPs with satisfactory stability and safety, making them suitable for therapeutic platforms, such as antibacterial and anticancer. The significance of this study lies in offering an eco-friendly, low-cost synthesis approach while expanding the applicability of safe Fe3O4-NPs for biomedical applications.
