A 14-year-old gelding in the Southeastern region of Australia was diagnosed with Mycobacterium ulcerans infection, initially presenting as a non-healing wound of the right flank progressing to extensive necrotic ulceration. Ziehl-Neelsen and Wade-Fite stained incisional biopsies revealed large numbers of intralesional acid-fast bacterial rods predominately within the areas of necrosis, supportive of Mycobacterium ulcerans infection. Real-time polymerase chain reaction (PCR) confirmed the presence of mycobacteria. The close relationship between M. ulcerans and M. marinum meant that PCR was unable to distinguish these species. Due to the equine host and non-aquatic environment there was a presumptive diagnosis of M. ulcerans. Multimodal treatment involved surgical debridement, biological debridement with maggots, cryotherapy, systemic antimicrobials and analgesia. The horse had made a full recovery at the time of publication. Infection with M. ulcerans needs to be considered in necrotizing soft-tissue lesions in regions where the organism is endemic, but it also needs further investigation due to the zoonotic nature of M. ulcerans with potential public health concerns.