The antibiotic overuse in hospitals, the food industry, and animal feed over past times has led to a significant rise in the incidence of antibiotic-resistant bacteria. To address these potentially life-threatening antibiotic-resistant illnesses, a quick identification and development of novel antimicrobials are necessary. The aim of this study was to synthesize a novel bacteriocin-nanoconjugates by combining selenium nanoparticles with purified bacteriocin from the Enterococcus faecium SMAA23 and investigate some of its biomedical activities. The nanoconjugates were characterized via X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray desorption (EDX), and zeta potential analytical techniques. There is investigation of the antibacterial, antifungal, and anticancer properties of nanoconjugates. Purified bacteriocin has a known molecular weight of approximately 43,000 Daltons. The characterization of nanoparticles and nanoconjugates was performed. The crystallite size of nanoconjugate was determined via X-ray diffraction (XRD) to be 15.29 nm. Transmission electron microscopy (TEM) detected particles of irregular form of nanoconjugate, measuring between 11 and 24 nm in diameter. Energy dispersive X-ray spectroscopy (EDX) confirmed the presence of selenium and protein. The measured zeta potential was - 12.1 + 0.12 mV. The results revealed potent antibacterial activity against Acinetobacter baumannii, with a growth inhibition zone of 23 mm ± SD. A minimum inhibitory concentration (MIC) of nanoconjugate was 15.625 µg/mL, while a minimum bactericidal concentration (MBC) was 31.25 µg/mL. The application of scanning electron microscopy (SEM) enhanced the rupture of the bacterial cell wall. The antifungal activity against C. albicans and C. tropicalis resulted in growth inhibition zones of 14 mm and 16 mm (± SD), respectively. Various concentrations of the nanoconjugate strongly inhibited MDA-MB-231 cells in the MTT experiment. The novel synthesized bacteriocin-nanoconjugates exhibited substantial antibacterial, antifungal, and anticancer properties.