Th e incidence of poxvirosis, a zoonosis caused by viruses of the family Poxviridae, has increased over the past two decades in Brazil. Th e important features of the Orthopoxvirus infection include rapid spread, occurrence restricted to lactating cows, lack of hygiene, person-to-person spread, and occurrence at properties where milking is conducted. We detected the presence of vaccinia virus antibodies, specifi cally for Cantagalo virus, in samples from two distinct populations from the state of Rio de Janeiro: (i) healthy professionals who live in the State of Rio de Janeiro and handle animals and (ii) in patients with cutaneous lesions compatible with poxvirosis. Th e plaque reduction neutralisation test (PRNT) was used. Of the 136 samples from the group of healthy professionals, 12 displayed seroreactivity with titres lower than or equal to 1/10. Anti-poxvirus antibodies were detected in 68.3% of samples of patients suspected to be infected with poxvirus (28/41). Th erefore, the plaque reduction neutralisation test constitutes a sensitive assay that can be more commonly used for diagnosis. Furthermore, professionals who handle animals should be included as an occupational risk group in areas with confi rmed human poxvirus cases.