Introduction: Due to the emergence of insecticide resistance among the vectors of human diseases, there is a need to explore the use of plant extracts which are safe alternatives to conventional chemical larvicides used in control of vector-borne diseases. The aim of this study was to evaluate the larvicidal activity of Acroptilon repens against third instar larvae of Anopheles stephensi, Culex pipiens and Culex quinquefaciatus. Materials and Methods: The aerial parts of A. repens plants was dried in the shaded places for 3 days at 26-28°C. The leaves, flowers and stems were blended to crashed form using an electric blender. The larvicidal activity of total extract of A. repens were evaluated against third instar larvae stage of mosquito vectors, An. stephensi, Cx. pipiens and Cx. quinquefaciatus under laboratory conditions with 24h exposure period. Data were subjected to probit regression analysis in order to estimate the lethal concentrations for 50% and 90% mortality values. Result: The extract of A. repens exhibited significant larvicidal activity against third instar larvae of An. stephensi, with 24h LC50 of 0.2970 and LC90 of 2.2097 mg/l. The LC50 and LC90 values were 2.5047 and 24.7374 mg/l for Cx. pipiens and 2.9047 and 16.1459 mg/l for Cx. quinquefaciatus. Conclusion: The extract of A. repens can serve as a natural larvicide against An. stephensi, Cx. pipiens and Cx.quinquefaciatus. According to the larvicidal properties of this plant, formulating an extract of A. repens which is known as an abundant agricultural weed in Iran creates an alternative to chemical larvicides and providing a job opportunities.