The use of botanical insecticides can serve as an alternative to the environmentally undesirable inorganic insecticides as a means of malaria vector control. The objective of the study was to identify the biologically active compounds of Oreosyce africana (Cucurbitaceae) leaf extracts by using a bioassay-guided approach on adult mosquitoes. The dichloromethane fraction of O. africana crude methanol extract was subjected to bioassay-guided fractionation by flash column chromatography, preparative thin-layer chromatography and high performance liquid chromatography to obtain purified fractions. The purified fractions were assayed against adult Anopheles arabiensis Patton. Gas chromatography coupled to mass spectrometry was used for identification of the active chemical compounds against adult mosquitoes. The LC50 and LC90 values were determined based on probit analysis. Two purified fractions of O. africana, fraction IV or B2’O and B2’’O, had the most potent adulticidal activities with an LC50 of 2.206 and LC90 of 7.811 ppm and with an LC50 of 2.62 and LC90 of 11.779 ppm at 24 hrs post-exposure, respectively. Among the purified fractions of O. africana LC50 ratio of B2’O and B2’’O showed significantly different values at P<0.05. The structural elucidation of the active ingredients using GC-MS analysis showed the presence of 9,12-Octadecadienoic acid (Z,Z) as the major chemical constituent (98.35%) in B2’O fraction and dibutyl phthalate as the major chemical constituent (97.75%) in B2’’O fraction with their molecular peaks at m/z 280 and 278, respectively. These results suggest that the purified fractions of O. africana possess a very high adulticidal effect against the principal malaria vector, An. arabiensis, in Ethiopia and have a potential for development into an affordable botanical mosquitocide as an alternative to the existing inorganic insecticides with environmental toxicity.