Abstract In permanent grassland the influence of NPK fertilizer addition and sward composition (proportion of plant functional groups) on soil microorganisms and the incorporation of 13C labelled glucose into microbial tissue were studied for 52 weeks. Microbial biomass was analysed by chloroform fumigation–extraction (CFE), soil microbial community structure by phospholipid fatty acids (PLFAs) and incorporation of labelled glucose into microorganisms by gas chromatography-combustion-isotope ratio mass spectrometry (GC–C–IRMS). 13C incorporation into PLFA biomarkers suggested that after two and six weeks fungi most efficiently captured glucose C compared to later sampling dates, whereas in bacteria it remained at a similar level for 52 weeks, suggesting that microbial residues are processed predominantly by bacteria not by fungi. Further the fungal-to-bacterial atom percent excess ratio of PLFA concentrations after two and six weeks exceeded one, indicating that in relative terms initially fungi are more efficient in capturing low molecular weight C compounds entering the soil in pulses than bacteria. NPK fertilizer decreased soil microbial biomass and changed microbial community structure but did not affect the fungal-to-bacterial PLFA ratio. In fertilized swards the fungal-to-bacterial 13C ratio increased, while bacterial 13C incorporation remained at the same level, suggesting that NPK fertilizer addition increases the efficiency of resource capture by fungi. Sward composition little affected microbial community composition and glucose C incorporation.