The Colorado potato beetle (CPB), Leptinotarsa decemlineata, poses a significant threat to potato crops globally and has developed resistance to numerous insecticides. Entomopathogenic nematodes (EPNs), from the genera Steinernema and Heterorhabditis, are promising biocontrol agents. This study aimed to identify the bacterial symbionts of ten native EPN isolates, evaluate the efficiency of cell free supernatants (CFSs) from their symbiotic bacteria against different developmental stages of CPB, and determine the effect of these CFSs on CPB developmental stages and lifespan. The recA gene region was utilized to determine the symbiotic bacteria of ten local EPNs. CFSs from these bacteria were applied orally and through contact to CPB’s various developmental stages (L1/L2, L3/L4 larval stages, and adults). Mortalities, developmental transition times, and lifespans of adults were observed. The CFSs showed significant toxicity to CPB, with higher efficiency against young larvae. The CFSs exhibited cumulatively lethal effects over time, particularly on L1/L2 larval stages. CFSs from X. bovienii exhibited the highest efficacy. In all cases, where larvae received CFSs orally or by contact, they failed to develop into pupae and adults. In contrast, the transition periods of old larvae to pupal and adult stages were comparable to those of the control group. Lifespans of adults differed based on bacterial isolates and application methods. This study shows the potential efficacy of CFSs from Xenorhabdus and Photorhabdus as biocontrol agents against CPB, particularly in its young larval stages. Further research is needed to unravel the mechanisms behind these effects and examine the impact on CPB mating and oviposition behaviors.