The overuse of antibiotics has induced the rapid development of antibiotic resistance in bacteria. As a result, antibiotic efficacy has become limited, and infection with multidrug-resistant bacteria is considered to be one of the largest global human health threats. Consequently, new, effective and safe antimicrobial agents need to be developed urgently. One promising candidate to address this requirement is selenium nanoparticles (Se NPs), which are made from the essential dietary trace element Se and have antimicrobial activity against Gram-positive bacteria. The size of nanomaterials can strongly affect their biophysical properties and functions; however, the effects of the size of Se NPs on their antibacterial efficacy has not been systematically investigated. Therefore, in this work, spherical Se NPs ranging from 43 to 205 nm in diameter were fabricated, and their mammalian cytotoxicity and antibacterial activity as a function of their size were systematically studied. The antibacterial activity of the Se NPs was shown to be strongly size dependent, with 81 nm Se NPs showing the maximal growth inhibition and killing effect of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). The Se NPs were shown to have multi-modal mechanisms of action that depended on their size, including depleting internal ATP, inducing ROS production, and disrupting membrane potential. All the Se NPs were non-toxic towards mammalian cells up to 25 μg mL-1. Furthermore, the MIC value for the 81 nm particles produced in this research is 16 ± 7 μg mL-1, significantly lower than previously reported MIC values for Se NPs. This data illustrates that Se NP size is a facile yet critical and previously underappreciated parameter that can be tailored for maximal antimicrobial efficacy. We have identified that using Se NPs with a size of 81 nm and concentration of 10 μg mL-1 shows promise as a safe and efficient way to kill S. aureus without damaging mammalian cells.