In the Lusatian mining district, rehabilitated mine soils contain substantial amounts of lignite in addition to recent carbon derived from plant litter. The aim of this study was to examine the importance of the two organic matter types as substrates for soil microbial biomass in mine soils containing organic matter with a contrasting degree of humification. Samples were taken from the lignite-containing overburden material, from a mine soil under 14-year-old black pine (Pinus nigra) and from a mine soil under 37-year-old red oak (Quercus rubra). Overburden material was ameliorated with alkaline ash and incubated in an identical manner as the 14-year-old and 37-year-old mine soils for 16 months. Carbon mineralisation was monitored throughout. After 0, 3, 6, 12 and 16 months, samples were removed and analysed for chemical parameters and for microbial biomass. In addition, C-14 activity measurements in bulk soil and microbial biomass were used to estimate their lignite content. Despite the high content of organic carbon in lignite-rich overburden material, low contents of microbial biomass were recorded. Ashamelioration led to high pH values in the overburden material, resulting in high concentrations of dissolved organic carbon most likely derived from lignite. Development of the microbial community was subsequently stimulated by presence of an easily available carbon source. In older mine soils, larger amounts of microbial biomass are most likely related to the presence of recent organic matter. Radiocarbon analysis of the microbial biomass extracted from the 14-year-old mine soil indicated higher lignite carbon contribution than recorded for microbial biomass of the 37-year-old mine soil. The highest concentration of lignite C present in microbial biomass as indicated by the C-mic/C-org ratio was, however, observed in the ameliorated overburden material. Therefore, we conclude that the importance of lignite as a carbon source for micro-organisms decreases when recent organic matter is present in the older stages of mine soil development.