The Saint Louis encephalitis virus (SLEV) is an encephalitogenic arbovirus (Flaviviridae family) that has a wide geographical distribution in the western hemisphere, especially in the Americas. The negevirus Brejeira (BREV) was isolated for the first time in Brazil in 2005. This study aimed to verify the existence of a possible interfering effect of BREV on the course of SLEV infection and vice versa. We used clone C6/36 cells. Three combinations of MOIs were used (SLEV 0.1 × BREV 1; SLEV 1 × BREV 0.1; SLEV 1 × BREV 1) in the kinetics of up to 7 days and then the techniques of indirect immunofluorescence (IFA), a plaque assay on Vero cells, and RT-PCR were performed. Our results showed that the cytopathic effect (CPE) caused by BREV was more pronounced than the CPE caused by SLEV. Results of IFA, the plaque assay, and RT-PCR showed the suppression of SLEV replication in the co-infection condition in all the MOI combinations used. The SLEV suppression was dose-dependent. Therefore, the ISV Brejeira can suppress SLEV replication in Aedes albopictus cells, but SLEV does not negatively interfere with BREV replication.