Assessment of soil microbial diversity, in terms of the actively growing population, may be a sensitive indicator of ecological stress and restoration processes. During this study, the relationship between microbial community structure; vegetation cover; and physical and chemical characteristics of topsoil covers at seven coal discard rehabilitation sites in South Africa were evaluated. A positive association was observed between the microbial biomass, vegetation cover and the organic carbon, ammonium, nitrate and phosphorus contents. Although the seven coal discard sites had different rehabilitation ages (1–8 years), there was no statistically significant difference between these sites based on physical and chemical characteristics. The various rehabilitation sites could be differentiated based on the microbial community structure. The Hendrina site was characterised by the highest viable biomass, monounsaturated fatty acids, and high ground cover. Bethal and Ermelo sites were the youngest sites (1 and 3 years) in terms rehabilitation age. Both these sites were associated with higher abundances of fungi and Actinomycetes than the other sites. Ratios of trans- to cis-monoenoic fatty acids and cyclopropyl fatty acids to their monoenoic precursors indicated stress in all sites sampled.