Background: Metabolic disruption and obesity are ongoing public health problems linked to chemical exposure. However, understanding causal pathways is difficult as real-world exposures occur as complex chemical mixtures rather than single chemicals. To improve understanding of the toxic effects of chemical mixtures, we obtained a set of real-world exposure mixture profiles derived from chemical biomonitoring data collected from the Environmental Influences on Child Health Outcomes Fetal Growth Studies (ECHO-FGS) birth cohort. The mixture profiles characterized five distinct exposure scenarios that consisted of varying observed concentrations of p,p’-DDE, PCB153, BDE47, and PFOS. While nuclear receptor signaling has been linked to metabolic dysregulation, mitochondria may also be a direct target of chemical exposure. These mixtures were evaluated for their effects on mitochondrial metabolism in two cell lines. The purpose of this work is to elucidate a mechanism how in utero exposure to mixtures of persistent pollutants may correlate to perturbed weight from birth through childhood. Methods: Observed concentrations of chemical mixture profiles of p,p’-DDE, PCB153, BDE47, and PFOS were examined at 1X, 50X, 250X, or 500X. Mixture profiles consisting of the four chemicals were tested individually, in chemical combinations, and as complete mixtures. We tested for mixture effects on metabolic disruption by examining whether the individual chemicals, chemical combinations or chemical mixtures perturb mitochondrial metabolism. Mitochondrial metabolism was measured as Oxygen Consumption Rate (OCR) after 1-hour or 24-hour exposures in HepG2 human hepatocarcinoma or C2C12 murine myoblast cells using MitoStress Tests on an Agilent Seahorse XFe24 Analyzer (Agilent Technologies, Santa Clara, CA). Results: MitoStress assays performed in HepG2 and C2C12 cells revealed that mixture Profiles 1, 3 and 4 perturb mitochondrial metabolism. Profile 3, dominated by BDE47, increased Basal Respiration, ATP-Coupled Respiration, Proton Leak, and Maximal Respiration greater than profiles 1 and 4 that are dominated by p,p’-DDE and PFOS, respectively. Interestingly, C2C12 cells were most sensitive to mixture exposures after only 1 hour; HepG2 cells were more sensitive after 24 hours. This suggests different mechanisms of action and a mechanism that is not dependent on transcriptional changes in C2C12 cells. PPAR activation has been hypothesized as a possible mode of obesogenic action; however, this is unlikely in C2C12 cells. Different combinations of chemicals were tested to determine the chemicals that might interact with BDE47. Chemical combinations revealed BDE47 negatively interacted with p,p’-DDE and PFOS. However, the dominant chemical typically drives the predominant effects measured on the mitochondria across profiles. Effects on the mitochondria were observed in multiple cell types and timepoints with greater sensitivity in C2C12 cells at serum-equivalent concentrations (1X) measured in mothers, indicating mitochondrial sensitivity. Conclusions: These findings highlight mitochondria as a potentially sensitive target of persistent organic pollutant mixtures. These mixtures perturbed mitochondrial metabolism in multiple cell types, concentrations and time points. Maternal serum equivalent concentrations produce significant short-term mitochondrial effects, highlighting the relevance of these exposures. Together, these results suggest that perturbation of mitochondrial function may be a mechanistic link between exposure to mixtures of persistent organic pollutants and metabolic dysregulation, potentially relevant to child health outcomes.