Botulism in cattle is rarely reported in Italy. This study describes an outbreak of botulism in a dairy herd in Central Italy in September 2012, and the notably high mortality rate it caused. Differential diagnoses involving toxicology and bacteriology, and electrolyte imbalances, all proved negative. A multiplex polymerase chain reaction (PCR) for detecting the BoNT gene led to the identification of the causative agent as Clostridium botulinum type DC. The presence of the toxin was confirmed subsequently via mouse bioassay. Initially, the peracute deaths and ambiguous clinical signs delayed the diagnosis and, as a result, impeded identification of the source of the infection on the farm. The severity of the outbreak demonstrates that screening for animal botulism should always form part of the diagnostic protocols used to investigate sudden peracute deaths without apparent cause in livestock.