FeOOH nanoparticles have recently appealed to wide-ranging applications due to their physicochemical properties and size-tunable synthesis; however, a few studies were performed on the antimicrobial potentials of iron oxyhydroxide nanoparticles. In this regard, we aimed to design various synthesis experiments to optimize the fabrication of β-FeOOH nanorods (NRs) with a desirable size of NRs and high antimicrobial potential. For this purpose, ten experiments were designed by manipulating reaction conditions of the standard hydrolysis method, including the initial concentration of ferric ions, reaction time, reaction temperature, and different concentrations of surfactants of PEI and PEG as process control agents. The structural characteristics of prepared NRs were analyzed using FE-SEM, FTIR, and XRD. The ImageJ software was also used to measure the length, width, and aspect ratio of NRs. Five microbial species, including the Gram-positive and Gram-negative bacteria and fungi species, were applied to investigate the antimicrobial potentials of NRs. The initial concentration of ferric ions revealed a dominant effect in NRs’ morphology, though other reaction conditions also played essential roles. The crystal structure of NRs was preserved in all synthesis experiments (β-phase) due to using the same iron salt precursors. The synthesized NRs exhibited dose-dependent antimicrobial activities against all tested microbial species. Additionally, the presence of surfactants exhibited an excellent capability of controlling effects on the size and growth pattern of NR crystals and improving their antimicrobial potentials; PEI could also be more effective on the antimicrobial efficacy of final NRs. Besides, our findings exhibited an inverse correlation between aspect ratio and antimicrobial potentials of β-FeOOH NRs. To sum up, it seems that optimization of synthesis conditions could provide tunable size and structure patterns of β-FeOOH NRs to achieve a promising tool for biomedical applications, particularly in combat with resistant microbial species, though further studies are needed in this regard.