Gastrointestinal mycobacteriosis in horses is difficult to diagnose because of the pathogen’s intracellular nature and the non-specific clinical symptoms. Effective accurate diagnosis facilitates prognosis and treatment. Current diagnostic procedures and methods of collecting material do not permit definitive antemortem diagnosis. However, culturing, acid-fast bacilli staining, histopathology, PCR and immunological marker evaluation may prove useful. Material and Methods: Three horses were admitted to a clinic for intensive care and a final diagnosis. Physical examination and additional tests were performed. Unfavourable prognoses and lack of treatment response prompted euthanasia decisions. Necropsy was performed, as were histological, microbiological and molecular investigations. Results: The clinical condition of the animals deteriorated despite therapy. Two horses were euthanised when they did not respond to treatment and had poor prognoses. Intestinal mycobacteriosis caused by Mycobacterium avium subsp. hominissuis was diagnosed postmortem using laboratory investigations. One horse’s diagnosis was established antemortem by cytological and microbiological examination of biopsy material from an abdominocentesis, and this animal was also euthanised because of its poor prognosis. Conclusion: Mycobacteriosis should be considered in the differential diagnosis of chronic debilitating equine diarrhoea in addition to rhodococcosis, lawsoniosis, salmonellosis, gastric ulcers and food intolerance. Peritoneal fluid obtained by abdominocentesis proved to be an effective diagnostic method for microbiological and molecular identification of Mycobacterium avium subsp. hominissuis in horses with suspected enteric mycobacteriosis and concomitant ascites