In recent years, the biosynthesized of metallic nanoparticles has been rapidly growing due to their environmentally synthesis and widespread applications in science and industry. In this study, we have reported the green synthesis of NiONPs using an aqueous extract of Trigonella subenervis (NiONPs@TS). The nanoparticles were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), and energy-dispersive X-ray spectroscopy (EDS). To find various applications for NiONPs@TS, the nanoparticles (NPs) were used as a catalyst for synthesizing some benzimidazole derivatives. The electrochemical behavior and antibacterial activity of NiONPs@TS were also evaluated. FE-SEM images exhibited a uniform spherical morphology in size of 28.21 nm for the biosynthesized nanoparticles. According to the XRD analysis, 26.43 nm was measured for NiONPs@TS crystal size. Benzimidazole derivatives were synthesized using NiONPs@TS as the catalyst with a yield of 69-92%. NiONPs@TS showed a significant electrocatalytic activity to glucose oxidation. The linear response range and detection limit were found to be 10-200 and 3.2 μM, respectively. Furthermore, NiONPs@TS exhibited an acceptable antibacterial activity in various assays, including well diffusion, disk diffusion, minimum inhibition concentration (MIC), and minimum bactericidal concentration (MBC). The best antibacterial activity was obtained against Bacillus subtilis with MIC of 4.0 ± 0.0 μg/ml and MBC of 6.7 ± 2.3 μg/ml. The obtained results showed the ability of NiONPs@TS in different fields such as catalytic, electrochemical sensing, and antibacterial activity, which can be contributed to the presence of biomolecules in T. subenervis extract.