Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an emerging zoonosis that has been associated with economic losses and social effects. Despite increasing reports of BV outbreaks in Brazil, little is known about the biological interactions of Brazilian VACV (VACV-BR) isolates during coinfections; furthermore, there are no tools for the diagnosis of these coinfections. In this study, a tool to co-detect two variants of VACV was developed to provide new information regarding the pathogenesis, virulence profile, and viral spread during coinfection with VACV-BR isolates. To test the quantitative polymerase chain reactions (qPCR) tool, groups of BALB/c mice were intranasally monoinfected with Pelotas virus 1—Group II (PV1-GII) and Pelotas virus 2—Group I (PV2-GI), or were coinfected with PV1-GII and PV2-GI. Clinical signs of the mice were evaluated and the viral load in lung and spleen were detected using simultaneous polymerase chain reactions (PCR) targeting the A56R (hemagglutinin) gene of VACV. The results showed that qPCR for the quantification of viral load in coinfection was efficient and highly sensitive. Coinfected mice presented more severe disease and a higher frequency of VACV detection in lung and spleen, when compared to monoinfected groups. This study is the first description of PV1 and PV2 pathogenicity during coinfection in mice, and provides a new method to detect VACV-BR coinfections.