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The problems of drug resistance in bacteria have become one of the daily challenges of the clinical treatment of patients, which inevitably forces us to use agents other than common antibiotics. Among these, we can take help from different properties and applications of nanoparticles (NPs). In this work, we evaluate the antibacterial activity of biosynthesized selenium nanoparticles (SeNPs) against standard strains of multidrug‐resistant Pseudomonas aeruginosa and Acinetobacter baumannii. The production of biosynthesized SeNPs was proved by ultraviolet‐visible, Fourier transform infrared, X‐ray diffractometer, Field Emission Scanning Electron Microscopy, Dynamic light scattering, and Zeta potential methods. The cytotoxicity effect of SeNPs was investigated by MTT assay. Disk diffusion agar (DDA) and minimum inhibitory concentration (MIC) tests were performed on the mentioned bacteria using different classes of standard antibiotics and SeNPs separately. The impact of SeNPs combined with the desired antibiotics for better treatment of these infections was evaluated by checkerboard assay to determine the synergism effect. After the confirmation results based on the biosynthesis of SeNPs, both standard bacterial strains were susceptible to SeNPs and had a zone of inhibition using the DDA test. Also, the results of MICs showed that biosynthesized SeNPs in lower concentrations than antibiotics cause no growth of bacteria. On the other hand, according to the checkerboard assay, SeNPs had a synergistic effect with conventional antibiotics. The antibacterial sensitivity tests demonstrated the inhibition of bacterial growth in the presence of lower concentrations of SeNPs than common antibiotics. This property can be exerted in future applications to solve the drug resistance obstacle of microorganisms in bacterial diseases. [ABSTRACT FROM AUTHOR]