Background: The management of bleeding originating from the guttural pouch (GP) has a high success rate, but the resolution of the macroscopic inflammatory lesions in the case of mycosis (GPM) is highly variable; the resolution of neurological disorders is inconstant and challenging. Objectives: Our aim was to test the feasibility and safety of topical oxygen therapy (TOT) in horses after induction of GPM and in cases with naturally occurring disease. Study design: This study was an in vivo experimental and retrospective two-phase study. Methods: During phase 1, the pilot study, both GPs were inoculated with Aspergillus fumigatus. One GP was randomly assigned to receive one to four TOT 30 min sessions with 100% medical oxygen at 9 L/min. Follow-up endoscopic images were assessed for scoring macroscopic inflammatory lesions of the pharynx and both GPs. In phase 2, the clinical study, TOT was administered for 45 to 60 min at 15 L/min in six horses presenting with GPM. Results: In phase 1, TOT administration was easy to perform in the standing horse with no adverse effects. After more than two administrations, macroscopic inflammatory lesions decreased more quickly in size in the treated GP. In phase 2, horses were treated with TOT only (n = 1) or combined with a transarterial coil embolization (TACE) procedure (n = 5). After TOT and discharge from the hospital, nasal discharge resolved in three horses, and improvement was noted in the fourth one. Between days 2 and 10 after admission, upper respiratory tract endoscopy (URTE) indicated size reduction and alteration in the appearance of all the macroscopic inflammatory lesions. The partial or total recovery of neurological disorders (2/4 laryngeal hemiparesis, 3/5 dysphagia, 1/2 dorsal displacement of the soft palate (DDSP), and 1/1 Horner's syndrome) was recorded. Main limitations: In phase 1, the small number of horses did not allow for statistically significant conclusions; in phase 2, clinical signs at admission varied between horses, which made comparison difficult. Conclusions: In adult horses, TOT alone or in combination with TACE is feasible and safe with a propensity to reverse the course and the progression of inflammatory lesions without additional local or systemic treatment.