These data comprise substrate utilisation profiles (using the BIOLOG gram-negative method) and moisture content data from soil sampled in an upland grassland experiment at Sourhope, Scotland. BIOLOG-GN (gram-negative) substrate utilisation analyses were used to give an indication of the ability of a subset of the bacterial community to utilise various carbon sources. These data include both temporal and spatial diversity in different depths of semi-natural grassland soil cores collected at different sample dates. Samples were collected in July 1999, October 1999, April 2000 and August 2000. Data were collected as part of the NERC Soil Biodiversity Thematic Programme, established in 1999 and centred upon the intensive study of a large field experiment located at the Macaulay Land Use Research Institute (now the James Hutton Institute)'s farm at Sourhope in the Scottish Borders (Grid reference: NT 8545 1963). During this time, the site was monitored to assess changes in aboveground biomass production (productivity), species composition and relative abundance (diversity). Full details about this dataset can be found at https://doi.org/10.5285/c42b0e3b-69e4-4941-addf-20c2e0612c58