The monkeypox virus (MPXV) outbreak, primarily endemic to Africa, has spread globally, with Brazil reporting the second-highest number of cases. The emergence of MPXV in non-endemic areas has raised concerns, particularly due to the co-circulation of other exanthematous viruses such as varicella-zoster virus (VZV) and molluscum contagiosum virus (MOCV). To perform an accurate differential diagnosis of MPXV during the ongoing outbreak in Minas Gerais, Brazil, a 5PLEX qPCR assay targeting orthopoxviruses (OPV), VZV, and MOCV was used to retrospectively analyze all clinical samples that tested negative for MPXV in the initial screening conducted at Funed. In summary, our study analyzed 1,175 clinical samples received from patients suspected of MPXV infection and found a positivity rate of 33.8% (397 samples) for MPXV using the non-variola qPCR assay. Testing the 778 MPXV-negative clinical samples using the 5PLEX qPCR assay revealed that 174 clinical samples (22.36%) tested positive for VZV. MOCV DNA was detected in 13 and other OPV in 3 clinical samples. The sequencing of randomly selected amplified clinical samples confirmed the initial molecular diagnosis. Analysis of patient profiles revealed a significant difference in the median age between groups testing positive for MPXV and VZV and a male predominance in MPXV cases. The geographic distribution of positive cases was concentrated in the most populous mesoregions of Minas Gerais state. This study highlights the challenges posed by emerging infectious diseases. It emphasizes the importance of epidemiological surveillance and accurate diagnosis in enabling timely responses for public health policies and appropriate medical care. Importance: Brazil ranks second in the number of cases during the global monkeypox epidemic. The study, conducted in Minas Gerais, the second most populous state in Brazil with over 20 million inhabitants, utilized differential diagnostics, revealing a significant number of positive cases for other exanthematous viruses and emphasizing the need for accurate diagnoses. During the study, we were able to assess the co-circulation of other viruses alongside monkeypox, including varicella-zoster virus, molluscum contagiosum virus, and other orthopoxviruses. The significance of the research is underscored by the concentration of positive cases in populous areas, highlighting the challenges posed by emerging infectious diseases. This demographic context further amplifies the importance of the research in guiding public health policies and medical interventions, given the substantial population at risk. The study not only addresses a global concern but also holds critical implications for a state with such a large population and geographic expanse within Brazil. Overall, the study emphasizes the pivotal role of surveillance and precise diagnosis in guiding effective public health responses and ensuring appropriate medical interventions.