Biotic stress is a major constraint affecting crops and their yield globally. Meloidogyne incognita, the root-knot nematode (RKN), is a harmful plant-parasitic nematode that is a major concern worldwide because of its impact on crop yield and quality. Additionally, the use of chemical nematicides has negative effects on beneficial soil microbes and crop quality and raises serious environmental concerns. Entomopathogenic nematodes (EPN) are seen as promising agents of biological control since they have demonstrated their efficacy in controlling insect pests, while research showed that they are also good at regulating nematodes. Three isolates of EPN species, Steinernema siamkayai (BSBT_S1), S. pakistanense (BSBT_S10) and Heterorhabditis bacteriophora (BSBT_H1), isolated from the semi-arid area of Rajasthan, were evaluated for their antagonistic activity against infective second-stage juveniles and gall hatching of M. incognita. When applied at different concentrations, H. bacteriophora was shown to have the strongest nematicidal activity compared to S. siamkayai and S. pakistanense in the laboratory experiments that revealed a reduction in juvenile population and gall hatching. All of the results indicate that H. bacteriophora is more pathogenic than the other two species, and further field trials are needed before it can be readily implemented as a biological control agent against root-knot nematodes.