Carbon dioxide (CO₂) has been approved by the US Environmental Protection Agency as a new aquatic pesticide to control invasive Asian carps and other aquatic nuisance species in the United States. However, limited CO₂ toxicity data could make it challenging for resource managers to characterize the potential risk to nontarget species during CO₂ applications. The present study quantified the toxicity of CO₂ to 2 native riverine fishes, bluegill (Lepomis macrochirus) and fathead minnow (Pimephales promelas), using 12‐h continuous flow‐through CO₂ exposure at 5, 15, and 25 °C water temperatures. Resulting survival indicated that bluegill (median lethal concentration [LC50] range 91–140 mg/L CO₂) were more sensitive to CO₂ than fathead minnow (LC50 range 235–306 mg/L CO₂) across all water temperatures. Bluegill were also more sensitive to CO₂ at 5 °C (LC50 91 mg/L CO₂, 95% CI 85–96 mg/L CO₂) than at 25 °C (LC50 140 mg/L CO₂, 95% CI 135–146 mg/L CO₂). Fathead minnow showed an opposite response and were less sensitive at 5 °C (LC50 306 mg/L CO₂, 95% CI 286–327 mg/L CO₂) relative to 25 °C (LC50 235 mg/L CO₂, 95% CI 224–246 mg/L CO₂). Our results show that CO₂ toxicity can differ by species and water temperature. Data from the present study may inform decisions related to the use of CO₂ as a control tool. Environ Toxicol Chem 2020;39:2247–2255. Published 2020. This article is a U.S. government work and is in the public domain in the USA.