Bacteria are often found in polymicrobial communities. Some of them are beneficial while others might be harmful or even lethal. The use of non-specific antimicrobial is widespread as there is a lack of specific antimicrobials. Contact with the gram-positive Staphylococcus aureus (S. aureus) bacterium is not only spreading the diseases but also initiating the massive loss in health-cost. Therefore, developing novel materials with antibacterial and antibiofilm is of great interest. In the present study, the metal–organic framework, NH2-MIL-125 has been synthesized, and primarily investigated for the antibacterial and antibiofilm activity. The synthesized NH2-MIL-125 is well characterized by PXRD, N2 adsorption/desorption isotherm and SEM. The results demonstrate the formation of uniformly crystalline, smooth-surfaced, square plate-shaped nanostructure. Further, the antimicrobial efficiency of NH2-MIL-125 was evaluated by microdilution followed by AlamarBlue and colony counting methods for S. aureus and Escherichia coli (E. coli, gram-negative). NH2-MIL-125 has shown specific antibacterial against S. aureus, whereas no activity was observed against E. coli. Similarly, the antibiofilm activity of NH2-MIL-125 evaluated by crystal violet staining has shown activity against S. aureus only. Moreover, NH2-MIL-125 has shown no cytotoxicity on HepG2 cells. Altogether, these results suggest that NH2-MIL-125 is a promising MOF for the design of antibacterial and antibiofilm agent against S. aureus.