Following the coronavirus disease 2019 (COVID-19) epidemic peak in Ariano Irpino, Campania region (Italy), we tested cattle for the presence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) on a cattle farm at which, prior to the investigation, 13 of the 20 farmworkers showed COVID-19-like symptoms, and one of them died. Twenty-four cows were sampled to detect SARS-CoV-2. All nasal and rectal swabs and milk samples were negative for SARS-CoV-2 RNA. Of the 24 collected serum samples, 11 were positive for SARS-CoV-2 nucleocapsid protein, 14 were positive for SARS-CoV-2 spike protein, and 13 were positive for SARS-COV-2-neutralising antibodies; all samples were negative for Bovine Coronavirus (BCoV), another betacoronavirus. To our knowledge, this is the first report of natural serological evidence of SARS-CoV-2 infection in cattle. We hypothesise that this may be a case of reverse zoonosis. However, the role of cattle in SARS-CoV-2 infection and transmission seems to be negligible.