The present study describes the morphological and immunohistochemical characteristics of a case of diffuse seminoma in a 16-year-old male mixed-breed horse. According to the owner, the animal's left testicle had been gradually increasing in size over a period of 2 months. On palpation, the testicle had a firm consistency, with no sensitivity to digital pressure, was adhered to the scrotum and measuring 16 cm × 8 cm. In the ultrasound examination, it presented a heterogeneous texture and areas of hypoechogenic echogenicity without visualization of the mediastinum. Therefore, the bilateral orchiectomy was performed. After the surgical procedure, it was found that the affected testicle presented a firm mass measuring 9 cm × 7 cm × 3.5 cm. Histologically, a multilobulated, non-encapsulated and invasive tumour mass was found, which replaced the seminiferous tubules, consisting of polygonal cells arranged in a mantle that varied from cohesive to loosely cohesive, supported by a scarce fibrous stroma. In the immunohistochemical examination, the neoplastic cells showed positive immunolabelling for OCT4 and C-KIT. In this report, the physical examination combined with the ultrasonographic examination were fundamental to the therapeutic management of the case, and the final diagnosis was made after histopathological and immunohistochemical tests.