Background: Bisphenol A (BPA) and perfluorooctanoic acid (PFOA) are synthetic compounds widely utilized in industrial activities devoted to the production of daily life plastic, metal products, and packaging from which they are able to migrate to food and water. Due to their persistence in the environment, living organisms are chronically exposed to these pollutants. BPA and PFOA have adverse effects on tissues and organs. The aim of this study was to identify the molecular targets and biochemical mechanisms involved in their toxicity. Methods: HepG2 and HaCaT cells were treated with BPA or PFOA, and the trypan blue exclusion test and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay were performed to define the conditions for subsequent investigations. We conducted quantitative PCR and western blot analysis to evaluate the expression of proteins involved in nitric oxide (NO) signaling. Cell-based assays were carried out to evaluate reactive oxygen species (ROS) production, nitrite/nitrate (NOx) accumulation, 3-nitrotyrosine (3-NT) formation, and mitochondrial membrane potential (MMP) determination in treated cells. Results: HepG2 and HaCaT cells incubated for 24 h with subtoxic concentrations of BPA or PFOA (50 and 10 \muM, respectively) exhibited altered mRNA and protein expression levels of NO synthase isoforms, manganese superoxide dismutase, and cytochrome c. Treatment with PFOA led to activation of inducible NO synthase (NOS), a marker of nitrosative stress, accompanied by the increased production of ROS, NOx, and 3-NT and alterations of the MMP compared to controls. Conclusions: The results of this study indicate the major involvement of the NO signaling axis in the persistent alteration of cell redox homeostasis and mitochondrial dysfunction induced by BPA and PFOA, highlighting the specific role of PFOA in NOS regulation and induction of nitro-oxidative stress.