Corneal ulcers in dogs pose a significant challenge in veterinary ophthalmology, often leading to prolonged visual impairment and discomfort. This study aimed to assess the efficacy of adipose tissue-derived mesenchymal stromal cell (ASCs) secretome as a treatment for complicated corneal ulcers in dogs. Ten dogs with spontaneous chronic corneal epithelial defects, were treated with topical application of ASC secretome eye drops. Our results showed that secretome therapy facilitated complete healing of all corneal ulcers within 4 weeks, with an average healing time of 1.2 weeks. Notably, secretome treatment was effective even in cases that had previously failed to respond to conventional therapies. Clinical signs such as blepharospasm, conjunctival hyperemia, and photophobia were alleviated promptly following secretome administration. Secretome therapy was well-tolerated, with no adverse reactions reported, further supporting its safety profile. The findings suggest that ASC secretome represents a promising cell-free and minimally invasive therapeutic approach for the treatment of complicated corneal ulcers in dogs.