Dengue virus (DENV) is an RNA virus belonging to the Flaviviridae family, comprising four antigenically distinct serotypes. Dengue is the primary arthropod-transmitted virus globally, posing a significant public health challenge, especially in Brazil, where the largest outbreak of Zika virus (ZIKV) was also recorded in 2016. ZIKV shares genomic and structural similarities with DENV, and their co-circulation in Brazil provides evidence of co-infection. The innate immune response against DENV and ZIKV is mediated by pattern recognition receptors that initiate intracellular signaling, leading to antiviral or inflammatory responses. This study aims to better understand the innate immune response to ZIKV in macrophages previously infected with DENV. To achieve this, bone marrow cells from C57BL/6 mice were differentiated into macrophages (BMDMs) and independently infected with each of the four DENV serotypes for 12 h, followed by ZIKV infection for an additional 12 h. Twenty-four hours post-infection, macrophage activation markers CD86 were assessed using flow cytometry and fluorescence microscopy. Pro-inflammatory and antiviral gene expressions were evaluated by qPCR. IFN-β was found to be down-regulated in all analyzed groups. No differences in CD86 expression were observed in ZIKV-infected BMDMs previously infected with DENV, except for serotype 4, which showed an increase in both activation markers. Conversely, TNF-α and IL-1β were down-regulated compared to non-infected or only DENV4-infected cells, correlating with increased cell viability and decreased production of the cytokine TNF-α. Bioinformatic analysis suggested that the expression of both cytokines might be regulated by miRNAs, including miR-181a-5p, which is also up-regulated in the innate immune response. Taken together, the results indicated that co-infection with DENV serotype 4 and ZIKV in mice BMDMs increases the expression of CD86, promoting macrophage activation, but reduces the expression of pro-inflammatory genes TNF-α and IL-1β, indicating enhanced cell viability what can be modulated by miRNAs.