Remote, harsh conditions of the Southern Ocean challenge our ability to observe the region’s influence on the climate system. Southern Ocean air-sea CO2 flux estimates have significant uncertainty due to the reliance on limited ship-dependent observations in combination with satellite-based and interpolated data products. We utilize a new approach, making direct measurements of air-sea CO2, wind speed, and surface ocean properties on an Uncrewed Surface Vehicle (USV). In 2019 the USV completed the first autonomous circumnavigation of Antarctica providing hourly CO2 flux estimates. Using this unique data set to constrain potential error in different measurements and propagate those through the CO2 flux calculation, we find that different wind speed products and sampling frequencies have the largest impact on CO2 flux estimates with biases that range from -4% to +20%. These biases and poorly-constrained interannual variability could account for discrepancies between different approaches to estimating Southern Ocean CO2 uptake.