Streptococcus equisub.zooepidemicus(SEZ) is described as a commensal bacterium of several animal species, including humans. Growing evidence supports the potential role of SEZ in the onset and progression of severe clinical manifestations of diseases in horses and other animals. In the present communication, we describe the diagnostic procedure applied to characterize the streptococcal infections caused by a novel SEZ sequence type (ST525) in donkeys raised on a farm in Abruzzo, Italy. The diagnostic process began with anamnesis and anatomopathological analysis, which revealed a severe bacterial suppurative bronchopneumonia associated with systemic vascular damage and haemorrhages. Then, SEZ infection was confirmed by applying an integrative diagnostic strategy that included standard bacterial isolation techniques, analytical tools for bacteria identification (MALDI-TOF MS), and molecular analysis (qPCR). Furthermore, the application of the whole-genome sequencing approach helped us to identify the bacterial strains and the virulence factors involved in animal diseases. The novel SEZ-ST525 was identified in two cases of the disease. This new sequence type was isolated from the lung, liver, and spleen in Case 1, and from retropharyngeal lymph nodes in Case 2. Moreover, the presence of the virulence genemf2, a virulence factor carried by prophages inStreptococcus pyogenes, was also found for the first time in an SEZ strain. The results of the present study highlight the need to apply an integrated diagnostic approach for the identification and tracking of pathogenic strains of SEZ, shedding new light on the re-evaluation of these bacteria as a causative agent of disease in animals and humans.