The biosynthesis of selenium nanoparticles (SeNPs) is one of the methods used alongside other conventional methods for SeNP synthesis. In this research, we used the cell-free culture (CFC) of Limosilactobacillus fermentum for SeNP synthesis. We investigated the biosynthesis of SeNPs under various levels of temperature, pH, and Se4+ concentration and characterized the biosynthesized SeNPs using FE-SEM, energy-dispersive X-ray, Fourier transform infrared spectroscopy, X-ray diffraction, ultraviolet–visible spectroscopy (UV–Vis), and dynamic light scattering–zeta potential analyses to find nanoparticles with desirable properties. Also, the cellular toxicity of SeNPs against the MCF-7 cell line was analyzed. The scavenging activity of free radicals in CFC before and after SeNP synthesis was examined using the DPPH method. The selected SeNP has an average hydrodynamic radius of 92.52 nm and a polydispersity index of 0.134. This nanoparticle also has a mostly spherical shape, amorphous nature, and zeta potential of −32.2 mV. The toxicity of nanoparticles for MCF-7 was much lower than sodium selenite salt. It was also confirmed that during nanoparticle synthesis, the reducing ability of CFC significantly decreases. This research aimed to design a safe, cheap, and eco-friendly protocol for the biosynthesis of SeNPs using the CFC of Limosilactobacillus fermentum. As a result, SeNPs possess great potential for further exploration in the realm of biomedicine.