Cattle parasitic otitis caused by the nematode Rhabditis spp. is a serious health problem in Brazil, since it does not have an effective control. In vitro studies associating biological control and chemical control as an alternative method showed promising results. The objective of the present study was to evaluate the combined use of the fungus Duddingtonia flagrans (AC001), 10% dimethylsulfoxide and 1.87% Ivermectin for the in vivo control of Rhabditis spp., in naturally infected Gyr cattle. For this purpose, 48 animals, whose infection in both ears was diagnosed, were randomly assigned to 6 groups: Group 1 (Ivermectin 1.87%); Group 2 (10% dimethylsulfoxide); Group 3 (AC001); Group 4 (Ivermectin 1.87% + dimethylsulfoxide 10%); Group 5 (1.87% Ivermectin + AC001); Group 6 (10% dimethylsulfoxide + AC001). The treatments were performed in a single dose, in the right ears, with the left ears remaining untreated, as a control group. There was a significant reduction (p<0.01) in the recovery of nematodes in the treated groups in relation to the control, with the following best efficacies: Groups 1 and 2, 47% and 52.9%, respectively, seven days after treatment; Groups 3, 4 and 5, 47.8%, 48.6% and 36.7%, respectively, 14 days post-treatment; Group 6, 38.4%, 21 days post-treatment. It was concluded that the combination of chemical compounds and D. flagrans in a single application were effective for the in vivo control of Rhabditis spp. in naturally infected cattle.