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, and four days later from a harvested maize field in Northern Germany and investigated these samples by pyrolysis-field ionization mass spectrometry (Py-FIMS) and hot-water extraction. Before tillage, BD showed much more volatilised matter (VM) during pyrolysis, indicating an increased amount of SOM. The Py-FIMS mass spectra revealed distinct differences in relative ion intensities of undisturbed soil 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 a restricted availability of labile carbon and, possibly an inhibitory effect of sterols from digestates. Despite high spatial variability, 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 carbohydrates and peptides, indicating an enhanced microbial activity. SOM composition in CL was unaffected by tillage. In summary, combining all analyses data provided strong evidence for significant shortterm SOM changes due to tillage in fertilized soils.