Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are widely (co-)detected in food and known for their hepatotoxicity in humans. Still, their combined toxicity needs to be investigated, especially the impact on mitochondria. In our previous work, we examined the effect of short-term exposure to different doses of AFB1, FB1, and their binary mixture (MIX) on the bioenergetic status of HepG2 cells, a well-recognized in vitro model system for studying liver cell function. In the current work, we further investigated the (combined) effect of AFB1 and FB1 on the mitochondrial and glycolytic activity of HepG2 cells using Seahorse respirometry analysis and RNA transcriptome sequencing. The results showed that the co-exposure, especially at high doses, is more toxic due to a more inhibition of all parameters of mitochondrial respiration. However, FB1 contributes more to the MIX effects than AFB1. RNA transcriptome sequencing showed that the p53 signaling pathway, a major orchestrator of mitochondrial apoptosis, was differentially expressed. Moreover, the co-exposure significantly downregulated the genes encoding for Complexes I, II, III, and IV, representing the onset of the suppressed mitochondrial respiration in HepG2 cells.