Haematobia irritans, also known as the horn fly, is a "plague" that spreads rapidly among cattle herds, especially in the southeast of Brazil. The aim of this study was to evaluate the interaction and activity of nematophagous fungus Duddingtonia flagrans (AC001) on H. irritans (Diptera: Muscidae). The experiment was conducted using the nematophagous fungus (AC001), which is harmless to animals, humans, and the environment. At the beginning of the experimental trial, samples of adult H. irritans were collected manually, directly from the dorsal region of naturally infested cattle of the Nelore breed. The flies where divided into two groups: groups of adult flies treated with AC001 (treated group) and groups of flies that did not receive treatment (control group). During the trial, the experiment was monitored daily for five days and the results were recorded. The results showed that the AC001 fungal isolate grew, colonized, and consequently caused the death of the flies in the treated group, while in the control group, no interaction or growth was observed, and the flies remained alive. It was concluded that the fungus D. flagrans interacted with adult flies, taking into consideration a "possible attack" by chitinase enzymes, since the fungal isolate drew on the chitin contained in the exoskeletons of the insects. In addition, attention should be focused on new studies that can demonstrate that, in the future, biological control of the horn fly could be an effective and safe method when compared with other methods.