An unprecedented outbreak of Oropouche virus (OROV) is occurring in the Americas, characterized by thousands of confirmed cases and a wide geographical spread, including areas outside the Amazon Basin. Little is known about this neglected arbovirus regarding its pathophysiological aspects and potentially different transmission modes. This study describes the clinical course of a man who returned from a trip to Cuba and presented to our hospital 4 days after the onset of febrile symptoms. The patient was diagnosed with Oropouche fever and was followed for 177 days after the onset of symptoms. We performed a longitudinal investigation of the samples collected from several body sites (whole blood, serum, urine, and semen) with the aim of providing further insights into OROV infection dynamics, using the detection of antigenomic RNA as a marker of active viral replication. Clinical samples that were longitudinally collected over the course of OROV infection showed consistently higher amounts of antigenomic RNA compared to genomic RNA, even after viral clearance from serum. Moreover, our case study showed the persistence of OROV RNA in serum of less than 15 days from the onset of symptoms, as compared to up to one month in urine, three months in semen, and four months in whole blood. Our study suggests that Oropouche virus may persist in an actively replicating state in different body sites for long periods of time, with important implications for transmission dynamics. Furthermore, our results provide a diagnostic indication, suggesting that serum is inferior to both urine and whole blood as preferred diagnostic samples. Further studies are needed to determine the pathogenetic implications of these findings, as they have been derived from a single case and must be confirmed using a larger number of cases.