The biological synthesis of nanoparticles, due to their environmental and biomedical properties, has been of particular interest to scientists and physicians. Here, iron nanoparticles (FeNPs) were synthesized using Satureja hortensis essential oil. Then, the chemical, functional, and morphological properties of these nanoparticles were characterized by typical experiments such as Uv-Vis, FTIR, XRD, FE-SEM, PSA, zeta potential, EDX, and EDX mapping. The results indicated Fe nanoparticles’ formation with a cubic morphological structure and a particle size in the range of 9.3-27 nm. The antimicrobial effects of these nanoparticles were further evaluated using disc diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungal concentration (MFC) against two gram-positive bacterial strains (Staphylococcus aureus and Corynebacterium glutamicum), two gram-negative bacterial strains (Pseudomonas aeruginosa and Escherichia coli), and one fungus species Candida albicans. The results showed that green-synthesized Fe nanoparticles possessed higher antimicrobial properties than Satureja hortensis essential oil against selected pathogenic microorganisms, especially Gram-negative bacteria. Finally, the anticancer effect of these Fe nanoparticles was investigated on human cancer cells, K-562, and MCF-7, by the MTT assay. The results showed the anticancer effect of these nanoparticles against selected cell lines.