Infective diseases are becoming more popular, and managing them has become a great worry for humanity. A rubber nanocomposite based on nitrile butadiene rubber with graphene oxide (NBR/GO) and GO/nanoclay (montmorillonite, MMT) (NBR/GO/MMT) was fabricated by a simple technique, roll milling. The synergistic influence of the existence of GO, clay, and electron beam (EB)-radiation on the NBR nanocomposites was characterized using scanning electron microscopy (SEM), and Fourier-transform infrared (FT–IR) techniques to study the mechanical, thermal, and antibacterial properties. The antibacterial activity of the prepared rubber nanocomposites was estimated via the disk diffusion process against Gram-positive bacteria, Bacillus subtilis, Staphylococcus lentus, and Gram-negative bacteria ; Pseudomonas aeruginosa and Proteus mirabilis. The results demonstrated that the physico-mechanical performance was significantly reinforced by incorporating nano GO (6 phr) and clay with GO (3 phr/6 phr. NBR films have no antibacterial potential. GO increases the antibacterial efficiency of the NBR films. NBR/3% GO/3% clay film is the most effective in reducing bacterial growth and B. subtilis was the most sensitive bacteria for rubber treatments. The inhibition zone diameters of the un-irradiated and irradiated NBR/3% GO/3% clay films for B. subtilis were 18.03 ± 0.59 and 25.63 ± 0.98 mm, respectively. It could be concluded that because of its outstanding flexibility and human-body compatibility penetration, rubber in corporation with antimicrobial agents can be utilized for manufacturing medical and environmental products.