Case series summary The present report describes middle ear cholesteatoma in two cats and also the use of video-otoscopy and flushing to assist with the diagnosis. CT and video-otoscopic examination and flushing were performed in two cats, a 13-year-old mixed breed spayed female cat and a 1-year-old mixed breed male cat, with middle ear cholesteatomas. During the procedure, keratinous material from the middle ears was collected for histopathological evaluation, demonstrating findings consistent with cholesteatoma, and the middle ears were flushed extensively. Relevance and novel information There is little information about middle ear cholesteatoma in cats, and to the authors’ knowledge, there are no reports in cats investigating the use of video-otoscopy to aid in the diagnosis of aural cholesteatoma, and this report demonstrates that it can aid in the diagnosis of this condition in cats. In addition, one of the cats had a concurrent otic polyp, which has not been previously reported in cats with cholesteatoma. Additionally, this is the first report of cholesteatoma in a young cat. The access to the cholesteatoma material was via ventral bulla osteotomy in one cat and via external canal without video-otoscopy in the other. More information regarding cholesteatoma in cats will help identify potential similarities and differences of this condition in cats compared with humans and dogs.