The synthesis of chemogenic selenium nanoparticles (SeNPs) was carried out by reducing sodium selenite and ascorbic acid and properly characterized the nanoparticles to ascertain its physicochemical characteristics. X-ray diffraction (XRD) results indicated that the SeNPs were highly crystalline and hexagonal with low crystallite size, which showed its possible application in catalyst, drug delivery, and bioimaging. The scanning electron microscopy (SEM) revealed clear spherical nanoparticle SeNPs with an average size of 19.91 nm that makes them surface reactive and suitable as biomedical materials. Energy dispersive X-ray (EDX) technique was used to verify that the high purity of the SeNPs (selenium as the major element) contained oxygen, carbon, sodium, chlorine, and gold as impurities. The SeNPs tested showed a prominent dose related scavenging effect with an antioxidant activity developed in presence of DPPH in the concentration with a maximum scavenging (38.32%) at µg/mL. Investigations on hemolysis revealed that SeNPs also do not lysis red blood cells and thus has a good biocompatibility. SeNPs had MIC values on Salmonella enterica of 16 µg/mL, which indicates their antimicrobial activity. Moreover, SeNPs showed significant antibiofilm potential with Salmonella enterica and in particular in its association with tetracycline where a high degree of synergism was achieved leading to more than 80% of inhibition. The results of this study’s findings indicate the promising future of SeNPs as antimicrobial and anti-oxidant application, which could possibly be used to overcome bacterial resistance as well as to act as an enhancer to the effects of conventional antibiotics.