Peronosclerospora spp. are economically important downy mildew pathogens of maize. The application of biotic agents is an alternative for controlling downy mildew. Biological control agent of Trichoderma spp. has been known not only directly fight pathogens but also induce resistance, including rapid production of Reactive Oxygen Species (ROS). This study aims to identify T. asperellum AC.3 in inducing maize resistance against downy mildew through ROS production around infected area. There were three treatments in this study, namely filtrate culture, suspension, and control, where each treatment was tested on two varieties, namely JH37 and PulutUri. The results showed that ROS accumulation around infected area was greater in maize plants treated with T. asperellum AC.3 filtrate and suspension in both varieties. The ROS production as pathogen response was detected at 6 hours and maintained up to 12 hours. The ROS in the filtrate culture treatment of the JH37 variety had the largest area, 243 mm2, followed by the suspension treatment of the JH37 variety, then the filtrate and suspension treatment of the PulutUri variety. T. asperellum AC.3 triggered ROS production in maize infected by the downy mildew pathogen, therefore, maize plants could localize the infection of Peronosclerospora spp.