Bovine parasitic otitis poses challenges in diagnosis, treatment and involves various agents, such as bacteria, fungi, mites, and nematodes. This study focused on the nematodes and bacteria isolated from the auditory canals of dairy cattle. A total of twenty samples were collected from dairy cattle in two states of Brazil. The results showed that Metarhabditis freitasi and M. costai nematodes were identified in 75% of samples. Bacterial species from the ear, identified via mass spectrometry, revealed that different strains were present in 65% of the cattle. Mycoplasma spp. were identified in 45% of samples through molecular techniques. Gram-negative bacteria and Mycoplasma spp. were exclusively found in nematode-infected cattle. Furthermore, the bacteria exhibited resistance to multiple antimicrobial classes, and demonstrating multiresistance. Electron microscopy revealed biofilm aggregates on the cuticle of Metarhabditis spp., suggesting a potential role of these nematodes in bacterial migration and interaction with nervous tissue. Thirteen bacterial strains demonstrated biofilm formation ability, indicating their potential pathogenic role. This research highlights the persistent and complex nature of parasitic otitis, emphasizing the significant role of nematode-bacteria associations in its pathogenicity. The presence of resistant strains and biofilm formation underscores the challenges in managing the diagnosis and treatment of bovine parasitic otitis.