The spread of Aquatic Invasive Species (AIS) between the Great Lakes and Mississippi River Basin by way of the Chicago Area Waterway System (CAWS) is a pressing concern to resource managers in the Midwest region. Augmenting this spread are watercrafts traveling through the CAWS locks and dams. AIS are able to attach to boat hulls, equipment, or are present in the surrounding water during lock transfers. It has been proposed that chemically treating boats during lock transfers would be an effective way to reduce the spread of AIS. Of a range of treatments identified as candidates to do this, hot water and dissolved ozone ranked high as effective treatments causing the least amount of environmental impact. This study assessed the lethality of hot water and dissolved ozone, separately and in combination, on select AIS in a laboratory setting. Species were exposed to water temperatures ranging from 18 to 50°C, ozone concentrations ranging from 0 to 920 ORP (Oxidation-Reduction Potential), and exposure durations ranging from 10 to 60 min to find treatments capable of inducing 100% mortality. Results indicated that water at 45°C achieved 100% mortality in all species tested with a 10 min exposure. Ozone concentrations induced significant mortality to all species tested, particularly when combined with elevated temperatures.