The occurrence of antibiotic resistance on common bacterial agents and the need to use new generations of antibiotics have led to the use of various strategies for production. Taking inspiration from nature, using bio-imitation patterns, in addition to the low cost of production, is advantageous and highly accurate. In this research, we were able to control the temperature, shake, and synthesis time of the synthesis conditions of Bacillus megaterium bacteria as a model for the synthesis of magnetic iron nanoparticles and optimize the ratio of reducing salt to bacterial regenerating agents as well as the concentration of salt to create iron oxide nanoparticles with more favorable properties and produced with more antibacterial properties. Bacterial growth was investigated by changing the incubation times of pre-culture and overnight culture in the range of the logarithmic phase. The synthesis time, salt ratio, and concentration were optimized to achieve the size, charge, colloidal stability, and magnetic and antibacterial properties of nanoparticles. The amount of the effective substance produced by the bacteria was selected by measuring the amount of the active substance synthesized using the free radical reduction (DPPH) method. With the help of DPPH, the duration of the synthesis was determined to be one week. Characterizations such as UV-vis spectroscopy, FTIR, FESEM, X-ray, and scattering optical dynamics were performed and showed that the nanoparticles synthesized with a salt concentration of 80 mM and a bacterial suspension to salt ratio of 2:1 are smaller in size and have a light scattering index, a PDI index close to 0.1, and a greater amount of reducing salt used in the reaction during one week compared to other samples. Moreover, they had more antibacterial properties than the concentration of 100 mM. As a result, better characteristics and more antibacterial properties than common antibiotics were created on E. coli and Bacillus cereus.