Green synthesis sources for synthesizing metal oxide nanoparticles are an interesting and expanding research area due to their potential antibacterial applications. Generally, nanoparticles are prepared using different chemical and physical methods that yield toxic or harmful nano-scaled particles in addition to the high cost and complicated processing steps. The present study successfully biosynthesized manganese oxide nanoparticles (MnO NPs) by reducing Manganese sulfate (MnSO4.H2O) using the cell-free supernatant of Bacillus subtilis ATCC6633. The formation of MnO NPs was confirmed by UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), Zeta analysis and transmission electron microscope (TEM). The biosynthesized MnO NPs displayed two absorption peaks at 285 and 353 nm. FT-IR spectrum proved the existence of bacterial proteins during the biosynthesis of MnO NPs that might act as stabilizing agents. MnO NPs have a negative charge of -20.4 mV according to Zeta analysis. TEM micrographs showed the rod-shape of MnO NPs with lengths of 70 to 100 nm and diameters of 10 to 23 nm. MnO NPs had a bactericidal action against Bacillus cereus and Escherichia coli with zones of inhibition of 23 and 25 mm, respectively in addition to minimum inhibitory concentration values of 20 and 15 µg/ml, respectively. The obtained results highlighted the possibility of using MnO NPs as a strong antibacterial agent in different industrial and medical applications.