This investigation determined the response of soil microbial communities to enhanced UV-B radiation and disturbance in upland grassland. A factorial field experiment encompassing two levels of UV-B supplementation (simulating ambient and a 30% increase in stratospheric ozone) and two levels of disturbance (disturbed and undisturbed) was established at Buxton Climate Change Impacts Laboratory, Derbyshire, UK, and maintained for 7 years prior to sampling. Enhanced UV-B increased microbial utilization of carbohydrates, carboxylic acids, polymers and aromatic compounds present in Biolog® GN plates when inoculated with soils taken from disturbed plots, but did not affect carbon utilization of soil microbial communities associated with undisturbed plots (UV-B×Disturbance interaction, P<0.05 for each substrate type). UV-B treatment did not affect numbers of bacteria or fungi. Direct microscopic counts showed fewer bacteria in soil originating from disturbed plots than from undisturbed plots (Disturbance, P<0.001), although a greater number of culturable bacteria and fungi were isolated from disturbed than from undisturbed soils (Disturbance, P<0.001). No UV-B- or disturbance-related differences in protein, starch or urea hydrolysis were exhibited by bacterial isolates. UV-B treatment did not affect total plant biomass within undisturbed plots or the biomass of individual groupings of grasses, forbs and mosses. Per cent root length colonized by arbuscular mycorrhizal fungi (AMF) was not affected by enhanced UV-B radiation in the undisturbed plots. Neither AMF nor plant biomass was measured in disturbed plots. The key findings of this study show that UV-B-mediated alterations in carbon utilization occurred in soil microbial communities subjected to disturbance, but such changes were not observed in communities sampled from undisturbed grassland. Differences in the catabolic potential of microbial communities from disturbed grassland subjected to enhanced UV-B are probably related to plant-mediated changes in resource availability or quality.