Aims: This study envisaged the isolation and characterization of magnetite nanoparticles (MNPs) from magnetotactic bacteria (MTB) and the evaluation of their antibacterial efficacy. Methods and Results:MNPs were extracted from 20 motile but morphologically different MTB, and they were subjected to antibacterial activity assay. These MNPs were found to be highly effective against Vibrio cholerae. MTB17 was considered as the potent MTB strain based on the antibacterial activity. The MNPs of MTB17 were isolated and validated by UV-Visible spectroscopy, particle size analysis, FTIR analysis, and PXRD.Conclusions: Isolation and characterization of ~85 nm MNPs from MTB is reported, and it is highly active against all the gram-positive and gram-negative strains tested. Significance and Impact of the Study:This study focuses on a novel use of biogenic magnetite MNPs as an antibacterial agent, which can be further explored using in vivo studies.