Oropouche virus (OROV) is an emerging arbovirus that causes acute febrile illness in humans in tropical areas of Central and South America. In Brazil, it is responsible for about half a million cases since its discovery. It is considered one of the most prevalent arboviruses in the Northern region of Brazil affecting mainly cities and villages in the Amazon and Central regions of the country. It is an RNA virus transmitted to humans through mosquito bites, mainly Culicoides paraenses, which transmits the so-called Oropouche fever, clinical manifestations similar to other frequent arboviruses such as Dengue, Chikungunya fever, and Zika virus disease. Despite its considerable presence in the Brazilian territory, the actual incidence of OROV infection can be masked by other arboviruses due to the similarity of symptoms, which leads to underreporting of cases, omission of potential disease-specific clinical aggravations, and makes specialized treatment difficult. At present, there are no specific therapeutic or preventive approaches to combat the virus, besides there is few data on its pathogenicity. Moreover, its vector has a wide geographic distribution in South and Central America, which makes evident the considerable risk of OROV dissemination and the emergence of a major public health problem similar to what happened with Chikungunya in 2014 and also with Zika in 2016. The study and understanding of morphological and metabolic alterations, including the identification of potential biomarkers in infected C6/36 and Vero cells and possible mechanisms associated with OROV infection in these cell lines by high-resolution electrospray ionization mass spectrometry (ESI-HRMS) suggests that OROV infection induces morphological and metabolomic alterations in both mosquito and mammalian cells, mainly associated with intravesicular trafficking and lipid synthesis. This information can contribute to the establishment of new targets for viral control and also to the development of a specific diagnosis.