The current report describes an outbreak of vesicular disease affecting dairy cows in midwestern Brazil in which a coinfection with 2 poxviruses-Vaccinia virus (VACV) and a parapoxvirus-was demonstrated. Milking cows presented vesicles, painful reddish or whitish papules, and scabby proliferative lesions in the teats and udder, in a clinical course of approximately 10-21 days. Histologically, multifocal areas of moderate to severe acanthosis, spongiosis, hypergranulosis, and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers were observed in the epidermis. Rounded eosinophilic inclusion bodies were observed in the cytoplasm of epithelial cells of areas with acanthosis or necrosis. Moderate inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, and macrophages were observed in some dermal areas. Two people milking the affected cows developed lesions on the hands, painful papules which progressed to ulcerative and scabby lesions in 4-7 days. Electron microscopy of scabs from 1 cow revealed the concomitant presence of orthopoxvirus and parapoxvirus particles. Scabs from 2 cows were positive by polymerase chain reaction for the parapoxvirus B2L gene; 1 of the scabs was also positive for the VACV vgf gene. Nucleotide sequencing of the B2L amplicon revealed a similarity of 96-99% with Orf virus (ORFV) and lower identity with Pseudocowpox virus (92-95%) and Bovine papular stomatitis virus (85-86%). Nucleotide sequencing of a region of parapoxvirus DNA polymerase gene revealed a high similarity (98-100%) with ORFV sequences. Thus, an unusual coinfection with VACV and a parapoxvirus, likely ORFV, was demonstrated in the outbreak.