Stress caused by soil salinity and soil drought limits cotton productivity in China. To determine the tolerance levels of cotton, we assessed the effects of soil salinity and soil drought on the biochemical characteristics of the roots of two cotton cultivars (CCRI-44, salt-tolerant; Sumian 12, salt-sensitive). Specifically, we analyzed root biomass, fatty acid composition, antioxidative enzyme activity, lipid peroxidation, H+-ATPase and Ca2+-ATPase activities. The cotton root biomass of the two cultivars declined significantly under conditions of soil salinity, soil drought, and the two stressors combined. The antioxidant enzyme activity of the roots also decreased markedly, which caused lipid peroxidation to increase, and changed the composition of the fatty acid membrane. H+-ATPase, Ca2+-ATPase and antioxidant enzyme activity decreased more under the two stressors combined. However, H2O2 content and O2 - generation increased under the two stressors combined, compared to each stressor separately. Overall, the combination of soil salinity and drought has a greater inhibitory effect and more harmful impact on root growth than each stressor separately. The higher tolerance of CCRI-44 to soil salinity and drought stress than Sumian 12 might be explained by differences in cotton root antioxidative enzyme activity. The lipid peroxidation levels of cotton roots might represent an important biochemical trait for stress tolerance.