Phenols are classified as polar narcotics, which are thought to cause toxicity by non-specific mechanisms, possibly by disrupting membrane structure and function. Here we test three phenolic chemicals, phenol, 2,4-dichlorphenol and pentachlorophenol on embryo development, heartbeat rate and mitochondrial respiration in fathead minnow (Pimephales promelas). While these chemicals have been used on isolated mitochondria, they have not yet been used to verify respiration in intact embryos. Mitochondrial respiration in intact embryos was measured after optimizing the Seahorse XFe24 Extracellular Flux Analyzer. Heartbeat rate and mitochondrial respiration patterns of fathead minnow embryos at different developmental stages were also characterized. Exposures of embryos at developmental stage 20 occurred for 24 h with five concentrations of each phenolic compound ranging from 0.85 to 255 μM for phenol, 0.49 to 147 μM for 2,4-dichlorophenol and 0.3 to 90 μM for pentachlorophenol. Exposure to phenol at the concentrations tested had no effects on development, heartbeat or mitochondrial respiration. However, both 2,4-dichlorophenol and pentachlorophenol showed dose-dependent effects on development, heartbeat rate, and mitochondrial respiration, with the effects occurring at lower concentrations of pentachlorophenol, compared to 2,4-dichlorophenol, highlighting the higher toxicity of the more chlorinated phenols. Both 2,4-dichlorophenol and pentachlorophenol decreased basal mitochondrial respiration of embryos and ATP production. These results indicate that higher chlorinated phenolic chemicals cause developmental toxicity in fathead minnow embryos by decreasing mitochondrial respiration and heartbeat rate.