As in human medicine, in veterinary medicine, chronic wounds are often related to polymicrobial infections and the presence of a biofilm, which compromises the effectiveness of therapeutic approaches. In this study, a Lusitano mare presented a 21-day-old chronic wound that was only being treated with an antiseptic. A swab sample was collected, and three isolates of Staphylococcus aureus and one of Pseudomonas aeruginosa were isolated. S. aureus did not show resistance to a panel of antibiotics. However, the P. aeruginosa isolate showed a resistance profile to carbapenems and fluoroquinolones, which may suggest a cross-resistance between antiseptic and antibiotics, given that no antibiotic therapy was applied to the wound or the mare in the previous year. Further experiments were conducted to assess the ability of the isolates to form biofilms, and to ascertain their susceptibility to gentamicin. The results demonstrated that the isolates produced biofilms. Gentamicin at the minimum inhibitory concentration (MIC) and 10× MIC caused biofilm removal between 59.3% and 85.7%, with the highest removal percentage being obtained for the P. aeruginosa isolate (at 10× MIC concentration). This study reveals that an equine wound was colonized by antibiotic resistant bacteria, and that all the wound colonizers could form biofilms, demonstrating the relevance of an adequate diagnosis and treatment when there is a suspicion of a biofilm-infected wound. It also highlights the possibility of resistance transmission between animals, animals and humans, or animals and the environment.