Invasive fish can profoundly affect communities they invade. In Hawai‘i, invasive fishes have become established in many anchialine pools, threatening the persistence of resident invertebrates, including several endangered species. Tools to eradicate invasive fishes from these pools are lacking. This study tested the efficacy of carbon dioxide (CO2) gas diffused into anchialine pool water as a method to eradicate invasive Mozambique tilapia (Oreochromis mossambicus), guppies (Poecilia reticulata), and western mosquitofish (Gambusia affinis). We first conducted aquarium trials to identify how these fishes were affected by elevated CO2 and the concomitant reduction in pH. We then carried out field trials in pools containing these fish in one pool each at two national historical parks on the Island of Hawai‘i during July 2021–January 2022. We also developed environmental DNA (eDNA) protocols to detect fish that may have survived CO2 treatments. The effect of CO2 on fish behavior varied among species; at pH 5.3 (CO2 = 255 mg/L) for tilapia and 5.0 (CO2 = 488 mg/L) for tilapia, guppies, and mosquitofish, all generally lost their ability to swim, showed slow or no gill movement, and altered their position in the water column. No tilapia survived the trials (n = 4 and 6 individuals at pH 5.3 and 5.0, respectively). In contrast, 41.7% (n = 12) of adult guppies and 66.7% (n = 12) of adult mosquitofish survived treatment at pH 5.0. In the field we were unable to reduce anchialine pool water pH below 5.7. Regardless, we were able to eradicate tilapia from one pool over four sequential treatments. Post-treatment eDNA assessments supported visual surveys, confirming our results. We were not able to eradicate guppies and mosquitofish. Results from this study show that CO2 can be an effective tool for eradicating invasive tilapia from anchialine pools, and post-treatment eDNA assessments can provide managers with a method for evaluating the success of eradication efforts.