In this study, we propose to synthesize NPs using plant extract containing active biomedical components, with the goal of obtaining NPs that inherit the biomedical activities of the plant. Herein, we report the synthesis of manganese dioxide nanoparticles (VBLE-MnO 2 NPs) using the leaves extract of Viola betonicifolia , in which the biological active plant’s secondary metabolites function as both reducing and capping agents. The synthesized NPs were successfully characterized with different spectroscopic techniques. The antibacterial, antifungal, and biofilm inhibition properties of the synthesized VBLE-MnO 2 NPs were further explored against a variety of bacteria (Gram-positive and Gram-negative) and mycological species. Additionally, their antioxidant ability against linoleic acid peroxidation inhibition, cytobiocompatibility with hMSC cells, and cytotoxicity against MCF-7 cells were investigated compared to leaves extract and chemically synthesized manganese dioxide NPs (CH-MnO 2 NPs). The results were demonstrated that the synthesized VBLE-MnO 2 NPs presented excellent antibacterial, antifungal, and biofilm inhibition performance against all the tested microbial species compared to plant leaves extract and CH-MnO 2 NPs. Moreover, they also exhibited significant antioxidant potential, which was comparable to the external standard (ascorbic acid); however, it was higher than plant leaves extract and CH-MnO 2 NPs. Furthermore, the synthesized CH-MnO 2 NPs displayed good cytobiocompatibility with hMSC cells compared to CH-MnO 2 NPs. The enhanced antioxidant, antibacterial, antifungal, and biofilm inhibition efficacy as compared to CH-MnO 2 NPs might be attributed to the synergistic effect of the VBLE-MnO 2 NPs’ physical properties and the adsorbed biologically active phytomolecules from the leaves extract of V. betonicifolia on their surface. Thus, our study establishes a novel ecologically acceptable route for nanomaterials’ fabrication with increased and/or extra medicinal functions derived from their herbal origins.