The primary goal of this research was to evaluate the phytochemicals, including bioactive components of Ocimum americanum extracts and assess their larvicidal potential against 3rd and 4th instar larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. The aqueous extraction process on O. americanum provides higher yield (9.61 ± 0.11%) and a significant volume of phytochemicals (tannins, saponins, terpenoids, flavonoids, alkaloids, protein, and carbohydrate) than other solvents (methanol, ethanol, acetone, petroleum ether, and chloroform) extracts. Fourier-transform infrared spectroscopy and high-performance liquid chromatography analyses revealed about eight predominant phytochemicals/functional groups in O. americanum aqueous extract. Surprisingly, in gas chromatography-mass spectrometry analysis, about 30 bioactive components were found in the aqueous extract of O. americanum, the majority of which had previously been identified as medicinally valuable bioactive components. Furthermore, these bioactive components containing aqueous extract demonstrated significant larvicidal activity against 3rd and 4th instar larvae of A. aegypti, An. stephensi, and C. quinquefasciatus after 24 h of treatment at concentrations ranging from 35 to 50 mg mL −1 . Thus, their outstanding larvicidal potential on 3rd and 4th instar larvae, the phytochemicals present in the aqueous extract of O. americanum could be used to control mosquito duplication and vector-borne diseases.