This study evaluates the biocontrol efficacy of cajeput oil against *Anopheles stephensi* mosquitoes and its effects on non-target species. Cajeput oil, derived from *Melaleuca cajuputi* leaves, was found to exhibit larvicidal properties against second to fourth instar *An. stephensi* larvae within 24 hours post-treatment. The essential oil significantly reduced detoxifying enzymes in larvae, indicating a direct impact on their immune system. The bioassay results show that cajeput oil effectively affects the second, third, and fourth instar larvae of *An. stephensi*. Additionally, the study found that cajeput oil does not induce toxicity in non-target *Eudrilus eugeniae* earthworms, as indicated by histological studies. Phytochemical screening and GC-MS analysis revealed the presence of several major phytochemicals contributing to the mosquito larvicidal activity. The findings suggest that cajeput oil is a promising candidate for biological control of *An. stephensi*, a vector of malaria, without causing significant harm to non-target organisms.This study evaluates the biocontrol efficacy of cajeput oil against *Anopheles stephensi* mosquitoes and its effects on non-target species. Cajeput oil, derived from *Melaleuca cajuputi* leaves, was found to exhibit larvicidal properties against second to fourth instar *An. stephensi* larvae within 24 hours post-treatment. The essential oil significantly reduced detoxifying enzymes in larvae, indicating a direct impact on their immune system. The bioassay results show that cajeput oil effectively affects the second, third, and fourth instar larvae of *An. stephensi*. Additionally, the study found that cajeput oil does not induce toxicity in non-target *Eudrilus eugeniae* earthworms, as indicated by histological studies. Phytochemical screening and GC-MS analysis revealed the presence of several major phytochemicals contributing to the mosquito larvicidal activity. The findings suggest that cajeput oil is a promising candidate for biological control of *An. stephensi*, a vector of malaria, without causing significant harm to non-target organisms.