In this study, essential oils and waste hydrosols of leaves of Ocimum tenuiflorum in four different geographical locations were extracted by hydrodistillation method and using gas chromatography / mass spectrometry (GC/MS) for chemical composition analysis. All four essential oil samples contained the main components (E)‐β‐caryophyllene (27.8‐49.0%), trans‐β‐elemene (20.3‐37.1%) and eugenol (9.0‐44.0%). Three of the four hydrosol samples had eugenol in absolute content (94.5‐98.6%), while the remaining hydrosol sample had two main components, elemicin (77.8%) and eugenol (14.2%). Essential oils and hydrosols demonstrated larvicidal activities against four important disease‐transmitting mosquito species including Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Culex fuscocephala with 24‐h LC50 values in the range 15.42‐56.01 µg/mL and 53.88‐97.80 µg/mL for the essential oils and the hydrosols, respectively. Essential oils and hydrosols strongly inhibited the acetylcholinesterase (AChE) enzyme of electric eels with IC50 values in the range of 25.35‐107.19 µg/mL. Microemulsion (ME) can be considered as a sustainable pesticide formulation over 300 days and has improved larvicidal activity compared to free essential oil. The O. tenuiflorum in Vietnam can be considered a low‐cost source of eugenol, botanical pesticides that control disease‐transmitting mosquitoes, as well as having therapeutic potential to be further investigated.