Natural resource agencies need effective strategies to control the spread of aquatic invasive species (AIS) such as invasive fish, which can expand their range using rivers as hydrological pathways to access new areas. Lock and dam structures within major rivers are prospective locations to deploy techniques, such as carbon dioxide (CO₂) infusion into lock water, that could impede upstream AIS migration without disrupting vessel passage and lock operation. The current pesticide label for CO₂ in the United States allows injections of 100–150  mg/LCO₂ as a behavioral deterrent treatment for invasive carps. This research describes the first operationalizing and testing of a CO₂ injection and manifold distribution system at a 1,548,000-L navigation lock chamber on the Fox River near Kaukauna, Wisconsin, USA. Two chemical distribution manifolds located on the floor and wall of the chamber were independently tested to quantify mixing time, mixing homogeneity, injection efficiency, and operational power requirements under a range of operating parameters. Both manifold configurations were able to meet most performance benchmarks established during previous fish behavior studies. Certain limitations were exhibited and quantified for both manifold configurations in terms of mixing homogeneity and operational power. This research details the design and performance of CO2-to-water infusion systems that could be used to deter the spread of AIS at navigation pinch-points. These results may inform future CO₂ system designs and operating conditions to support natural resource management plans to limit the spread of AIS.