The present study reports a low-cost, eco-friendly, and reproducible microbes Lactobacillus sp. mediated biosynthesis magnesium oxide nanoparticles. The nanoparticles were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and high-resolution transmission electron microscopy (HRTEM). The morphological characteristics were found to be spherical, oval in shape, individual nanoparticles as well as a few aggregates. The XRD shows the crystallographic plane indicating that nanoparticles structure dominantly corresponds crystalline. The biosynthesized magnesium oxide nanoparticles showed corresponding functional peaks. The cytotoxic effects of the magnesium oxide nanoparticles could significantly inhibited HL-60cancer cell lines proliferation in a time and concentration-dependent manner by MTT assay. L. sporogenes mediated magnesium oxide nanoparticles had potential to inhibit the cancerous cells by 60% while L. plantarum mediated nanoparticles found to inhibit the growth by 50%. The biosynthesis of nanoparticles has been proposed as an environmental friendly and cost effective alternative to chemical and physical methods. Hence, this report added the value for the application of magnesium oxide nanoparticles in biomedical and nanotechnology applications with the absence of adverse side effects from nonpathogenic, mesophilic Lactobacillus plantarum and Lactobacillus sporogenes for the nanoparticles. © 2017, Springer Science+Business Media, LLC, part of Springer Nature.