In this work we studied the effect of Yellow Fever Virus (YFV) infection on mitochondrial network dynamics (MD). Using the A549 cell line as a model, we examined MD during YFV infection at day 1 post-infection (dpi), when only some cells are infected, and at day 3, when viral infection has spread. Although we did not detect significant differences in total mitochondrial mass using flow cytometry, confocal and MiNA image analysis at 1 dpi showed that YFV increased the number of individual structures, networks, and branches compared with mock-infected cells. In contrast, no differences were observed at 3 dpi. Using two different mitotimer plasmids, the results showed that YFV slightly increased mitochondrial biogenesis and mitophagy at 1 dpi. Confocal studies of Parkin 2 and F1-ATPase colocalization supported these results. Treatment of cells with CCCP, a reversible ionophore that triggers mitochondrial fragmentation, increased the number of individual structures and networks one day later and had opposite effects at three days after treatment. Treatment also decreased viral titers in cell supernatants. On the other hand, treatment of cells with Mdivi-1, an inhibitor of mitochondrial fission that leads to mitochondrial elongation, increased the number of individual structures one day later and decreased the number of individual structures, networks, and branches at three days after treatment, with no change in viral titers. As expected, YFV infection increased IFN-I transcription and PKR expression, but these levels were also modulated by the drugs CCCP and Mdivi-1. Our results suggest that YFV infection induces early changes at MD that affect viral replication and open new strategies for developing new treatments.