Nematode-trapping (NT) fungi are natural predators of the soil living nematodes. Diverse external signals mediate the generation of predatory devices of NT fungi. Among these, broad ascarosides and nitrogenous ammonia are highly efficient inducers for trap structure initiation. However, the overlay effect of ammonia and ascaroside on the trap morphogenesis remains unclear. This study demonstrated that the combination of nitrogenous substances with nematode-derived ascarosides led to higher trap production compared to the single inducing cues; notably, ammonia and Ascr#18 had the most synergistic effect on the trap in A. oligospora. Further, the deletion of ammonia transceptor Amt43 blocked trap formation against ammonia addition in A. oligospora but not for the ascaroside Ascr#18 induction. Moreover, ammonia addition could promote plasma endocytosis in the process of trap formation. In contrast, ascaroside addition would facilitate the stability of intracellular organization away from endocytosis. Therefore, there is a synergistic effect on trap induction from different nitrogenous and ascaroside signals.