Selenium nanoparticles (SeNPs) are increasingly recognized for their exceptional antibacterial properties. This study aimed to develop a green, safe, and efficient method for the biosynthesis of SeNPs using the fungus Eurotium cristatum, a novel approach in SeNP synthesis. The process yielded (36.40 ± 4.22) mg of SeNPs per liter of 1.2 mmol/L sodium selenite supplementation. These SeNPs exhibited an average diameter of 231.7 nm and a negative charge, and they remained stable when stored at 4 ℃ . Ultraviolet and visible spectrophotometry revealed a maximum absorption peak at 212 nm, suggesting effective nanoparticle formation. Fourier transform infrared spectrometry indicated that proteins and carbohydrates in the mycelium contributed to the SeNP synthesis. Concentrations of SeNPs below 50 μg Se/mL did not exhibit cytotoxic effects on the growth and proliferation of human hepatocyte L-02 cells. The minimum inhibitory concentration of SeNPs was found to be 2 mg/mL against both methicillin-resistant Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). The SeNPs compromised the cellular integrity of test strains, causing leakage of intracellular contents and disruption of the oxidative stress system, leading to irreversible damage. Our results demonstrate the potential of SeNPs biosynthesized by E. cristatum to act as effective antibacterial agents, signifying a novel and promising approach to developing natural antimicrobial solutions.