Biosynthesis has gained growing interest due to its energy efficiency and environmentally benign nature. Recently, biogenic iron sulfide nanoparticles (FeS NPs) have exhibited excellent performance in environmental remediation and energy recovery applications. However, their biosynthesis regulation strategy and application prospects in the biomedical field remain to be explored. Herein, biogenic FeS NPs are controllably synthesized by Shewanella oneidensis MR-1 and applied for cancer therapy. Tuning the synthesis rate and yield of biogenic FeS NPs is realized by altering the initial iron precursor dosage. Notably, increasing the precursor concentration decreases and delays FeS NP biosynthesis. The biogenic FeS NPs (30 nm) are homogeneously anchored on the cell surface of S. oneidensis MR-1. Moreover, the good hydrophilic nature and outstanding Fenton properties of the as-prepared FeS NPs endow them with good cancer therapy performance. The intracellular location of the FeS NPs taken up is visualized with a soft X-ray microscope (SXM). Highly efficient cancer cell killing can be achieved at extremely low concentrations (<12 μg mL-1), lower than those in reported works. Such good performance is attributed to the Fe2+ release, elevated ROS, reduced glutathione (GSH) consumption, and lipid hydroperoxide (LPO) generation. The resulting FeS NPs show excellent in vivo therapeutic performance. This work provides a facile, eco-friendly, and scalable approach to produce nanomedicine, demonstrating the potential of biogenic nanoparticles for use in cancer therapy.