This study investigates the potential of entomopathogenic nematodes (EPNs) as biological control agents by exploring their occurrence and diversity in Amazcala, Querétaro. The aim was to characterise their distribution and evaluate their pathogenicity against insect pests. Soil samples were collected from various agricultural lands, followed by laboratory isolation and the molecular identification of EPN species. Morphological and genetic analyses confirmed the presence of several species with distinct pathogenic profiles. Pathogenicity assays using the larval stages of Galleria mellonella and Tenebrio molitor revealed that Heterorhabditis bacteriophora and Heterorhabditis atacamensis exhibited significant virulence, with Galleria mellonella being more susceptible. Among the 12 recovered EPN isolates, three strains—AMZX05 (Heterorhabditis atacamensis), AMZX10 (Heterorhabditis bacteriophora), and AMZX13 (Heterorhabditis atacamensis)—demonstrated particularly high pathogenic potential. These strains represent promising candidates for biological control and could contribute to sustainable integrated pest management (IPM) strategies. Further research is recommended to optimise their application across diverse agroecosystems.