Abstract. Tillage induces decomposition and mineralisation of soil organic matter (SOM) by the disruption of macroaggregates and may increase soil CO₂ efflux by respiration, but these processes are not well understood at the molecular level. We sampled three treatments (mineral fertiliser: MF; biogas digestate: BD; unfertilised control: CL) of a Stagnic Luvisol a few hours before and directly after tillage as well as 4 days later from a harvested maize field in northern Germany and investigated these samples by means of pyrolysis-field ionisation mass spectrometry (Py-FIMS) and hot-water extraction. Before tillage, the Py-FIMS mass spectra revealed differences in relative ion intensities of MF and CL compared to BD most likely attributable to the cattle manure used for the biogas feedstock and to relative enrichments during anaerobic fermentation. After tillage, the CO₂ effluxes were increased in all treatments, but this increase was less pronounced in BD. We explain this by restricted availability of readily biodegradable carbon compounds and possibly an inhibitory effect of sterols from digestates. Significant changes in SOM composition were observed following tillage. In particular, lignin decomposition and increased proportions of N-containing compounds were detected in BD. In MF, lipid proportions increased at the expense of ammonia, ammonium, carbohydrates and peptides, indicating enhanced microbial activity. SOM composition in CL was unaffected by tillage. Our analyses provide strong evidence for significant short-term SOM changes due to tillage in fertilised soils.