The biosynthesis of materials using medicinal plants can be a low-cost and eco-friendly approach due to their extraordinary properties. Herein, we reported a facile synthesis of Fe3O4 nanoparticles using Malva sylvestris. The surface morphology, functional groups, and elemental analysis were done to characterize the synthesized nanoparticles. The cytotoxicity performance of the synthesized nanoparticles was analyzed by exposing nanoparticles to MCF-7 and Hep-G2 cancer cell lines through MTT colorimetric assay and the IC50 value was defined as 100 μg/mL and 200 μg/mL, respectively. The antibacterial performance of synthesized nanoparticles against four different bacterial strains including Staphylococcus aureus, Corynebacterium, Pseudomonas aeruginosa, and Klebsiella pneumoniae were assessed through microdilution broth method. The synthesized Fe3O4 nanoparticles using Malva sylvestris demonstrated higher antibacterial effects against Gram-positive strains with MIC values of 62.5 μg/mL and 125 μg/mL which increase the inhibitory percentage to more than 90%.