Luminescent bacteria, having reductase enzymes, can reduce metal ions as nanoparticles. In this study, the synergic effect of biosynthesized selenium nanoparticles (SeNPs), using Pseudomonas putida KT2440, carrying lux AB gene with biogenic gold nanoparticles (AuNPs) is assessed. The SeNPs were first purified and then characterized using different techniques, including UV-visible and Fourier Transform Infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Energy Dispersive x-ray (EDX) spectroscopy analyses. The antibacterial and antibiofilm activities of SeNPs were also evaluated with and without AuNPs, at 12.5, 25, 50, and 100 mg l−1 concentrations. The SEM images showed that biogenic SeNPs were spherical attached to the surface of the producing bacteria. The FTIR spectroscopic results revealed the presence of protein molecules on the surface of the synthesized SeNPs. It is found that purified SeNPs have proper antibacterial and antibiofilm activities against several potential human pathogenic bacteria, depending on the NPs concentrations. It was also found that the presence of AuNPs enhances the antibacterial activity of SeNPs. Our results showed that adding SeNPs improved the antibacterial activity of AuNPs against Bacillus subtilis (71%), Staphylococcus aureus (55%), Staphylococcus epidermidis (52%), Escherichia coli (47%), and Pseudomonas aeruginosa (26%). Also, the antibiofilm activity of SeNPs improved by about 26% and 37% against E. faecalis and S. aureus, respectively. In conclusion, the mixture of biosynthesized SeNPs and AuNPs, using luminescent bacteria as Se-Au nanoparticles is more effective and can be applied as an antimicrobial agent against multi-antibiotic resistant pathogenic bacteria.