The potential application of biological copper (Cu) nanoparticles (BCuNPs) as fungicides, insecticides, and fertilizers has piqued the interest of agricultural scientists. In the current study, BCuNPs were biosynthesized by an isolate of Bacillus, which was determined to be Bacillus subtilis subsp. subtilis. This strain can tolerate copper concentrations up to 12 mM and reduce copper by synthesizing BCuNPs. Ultraviolet-visible spectroscopy, dynamic light scattering (DLS), Zeta potential, Fourier transform infrared (FTIR), and transmission electron microscopy (TEM) equipment were used to characterize BCuNPs. As assessed by TEM and DLS, they ranged in size from 15 to 45 nm, with an average particle size of 23 nm. It was necessary to conduct both in vitro and in vivo studies to confirm the nematicidal activity of the fabricated BCuNPs on the infective second-stage juveniles (J2s) and eggs ofMeloidogyne incognita the causal agent of root knot disease of cucumber. The in vitro application of different concentrations (100, 125, 150, 175, 200, 225, and 250 ppm) of BCuNPs significantly (P<0.05) increased the number of dead (J2s) of M. incognita, and significantly (P<0.05) decreased the percentage of egg hatching of M. incognita after 12, 24, 48, 72, and 96 h of application compared to the negative control (distilled water) with 250 ppm being the most effective dose of application after 96 h of application. Furthermore, the in vitro application of these different concentrations of BCuNPs combined with nematicide Tervigo® (abamectin, at the recommended application rate (RAR) of 0.1), significantly (P<0.05) increased the number of dead (J2s), and significantly (P<0.05) decreased the percentage of egg hatching of M. incognita compared to the negative control (distilled water), the positive control (Tervigo® alone), and compared to the BCuNPs alone, with 250 ppm being the most effective dose of application after 96 h of application. The number of galls, number of egg masses, root gall index, egg mass index of M. incognita on infected cucumber, the population density of M. incognita J2s 100 g soil-1, as well as the population of Tylenchorhynchus spp., Pratylenchus spp., and Helicotylenchus spp. in soil, were all significantly (P<0.05) reduced by 100, 150, 200, and 250 ppm of BCuNPs when applied under greenhouse conditions, with 250 ppm being the most toxic compared to the untreated of M. incognita treatment. When the nematicide abamectin (used at 0.1 RAR) and BCuNPs (used at 250 ppm) were used together in vivo, they had a synergistic effect on M. incognita. This treatment was found to be significantly (P<0.05) better than the nematicide abamectin alone in reducing the number of galls, the number of egg masses, the root gall index, the egg mass index of M. incognita on infected cucumber, and the population density of J2s 100 g soil-1. These findings indicate that the use of BCuNPs at 250 ppm either individually or combined with nematicide abamectin in integrated pest management systems for controlling nematodes infesting cucumber is recommended