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The McMurdo Dry Valleys of Antarctica are a climate-sensitive ecosystem, where future projected climate warming will increase liquid water availability to release soil biology from physical limitations and alter ecosystem processes. For example, many studies have shown that CO2 flux, an important aspect of the carbon cycle, is controlled by temperature and moisture, which often overwhelm biotic contributions in desert ecosystems. However, these studies used either single-point measurements during peak times of biological activity or diel cycles at individual locations. Here, we present diel cycles of CO2 flux from a range of soil moisture conditions and a variety of locations and habitats to determine how diel cycles of CO2 flux vary across gradients of wet-to-dry soil and whether the water source influences the diel cycle of moist soil. Soil temperature, water content and microbial biomass significantly influenced CO2 flux. Soil temperature explained most of the variation. Soil CO2 flux moderately increased with microbial biomass, demonstrating a sometimes small but significant role of biological fluxes. Our results show that over gradients of soil moisture, both geochemical and biological fluxes contribute to soil CO2 flux, and physical factors must be considered when estimating biological CO2 flux in systems with low microbial biomass.