An outbreak of strangle-like disease involving 26 horses farmed in central Italy was investigated by clinic examination, endoscopy, cytology, bacteriology and polymerase chain reaction (PCR). At weekly interval, a total of three nasal swabs and one guttural pouches lavage fluid (GPLF) were collected, and no Streptococcus equi subsp. equi carrier was found. Some horses showed upper airways disease and endoscopic signs of pharyngeal lymphoid hyperplasia of different grade and/or abnormal endoscopic appearance of guttural pouches. Streptococcus dysgalactiae subsp. equisimilis was isolated from 14 horses while S. equi subsp. zooepidemicus was isolated from six horses. PCR confirmed the biochemical and serological identification of all isolates and was positive in 10 bacteriological negative samples. The absence of S. equi and the frequent detection of S. equisimilis and S. zooepidemicus suggest that beta-haemolytic streptococci other than S. equi could be the causative agent of strangle-like disease.