Microbial pollution of water is one of the hazardous factors that threaten public health and the surrounding ecosystem. Therefore, the development of environmentally benign nanoparticles with exceptional properties has increased the drive toward improvement of water/wastewater disinfection. Accordingly, we aim to address the antagonistic effect of biogenic hematite nanoparticles (HNPs) against a broad spectrum of microorganisms and in water/wastewater disinfection systems. The minimum inhibitory concentration of HNPs at a range of 0.075–9.6 mg mL−1 is examined. The results demonstrate that Gram‐positive bacteria are more susceptible to HNPs than Gram‐negative ones. In addition, the growth of Candida albicans and Aspergillus brasiliensis is inhibited by applying 1.2 mg mL−1 of HNPs, which indicates antifungal activity. Moreover, HNPs (0.3 mg mL−1) inhibits 58.7, 50.5, and 86.3% of the growth of Staphylococcus aureus, Pseudomonas aeruginosa biofilms, and Chlorella vulgaris, respectively. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) of untreated and HNPs‐treated cells of Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Candida albicans, and Chlorella vulgaris are studied, and the ultrastructure micrographs show ultrastructure deformities caused by HNPs. Remarkable disinfection strength reaches up to 99.8%, mainly with increasing dose and contact time. The current study highlights the efficiency of HNPs as a biocide, which could contribute to water/wastewater treatment as an alternative for chlorine.