Background: Natural products derived from plants and secondary metabolites from microorganism can promise for the discovery of synthetic analogs with improved efficacy, potency, and safety. Our study attempted to study the effect of new formula as mosquito larvicide. Methods: Isolation and characterization of Prodigiosin and essential oil from Thuja orientalis and purification of PDG. Investigating the dose response bioassay, the synergistic effect, and the mode of action for each preparation. Results: The treatment of the 3rd larva stage of Cx. pipiens revealed that LC₅₀ of PDG and T. orientalis leaves’ E.O were (39.5 ± 0.341 ppm &102.9 ± 0.46 ppm respectively) after 24 hours. The combination of LC10 of PDG with LC25 & LC50 of the E.O. showed a synergistic effect resulting in 33.3% and 100% of death, respectively. Individual and combination treatment showed reduction in the activity of acetylcholine esterase, total protein and AChE specific gravity as compared to untreated 3rd larva stage of Cx. pipiens. PDG and E.O. resulted in reduction in midgut pH leading to cellular respiration inhibition as compared to untreated larvae that showed alkaline medium. Conclusions: So PDG and the T. orientails leaves’ oil combination showed a promising synergistic potency against the 3rd larva stage of Cx. pipiens.