To investigate how the chemical composition of native organic matter of two contrasting soils varies with inputs of biochar and fresh material (including plant roots) and how these underlying changes influence microbial community structure. Corn stover (CS) and CS-derived biochars produced at 350 °C and 550 °C were applied at a dose of 7.2 t C ha−1 to two contrasting soils—an Alfisol and an Andisol. After 295 days of incubation, two undisturbed subsamples from each pot were taken: (i) in one, lucerne (Medicago sativa L.) was seeded (plant study, P) and (ii) in the other, the incubation was continued without the plants (respiration study, R); all subsamples were incubated for an additional 215 days. Soils without amendments were used as controls. At the end of the incubation (510 days), their bacterial community profiles were characterised using ARISA and the molecular composition of soil organic matter (SOM) was investigated by pyrolysis-GC/MS. There were significant interactions between soil type, study type (P or R) and organic amendment. Organic amendments influenced overall SOM composition with microbial community response being mainly influenced by soil type but also strongly affected by the presence or absence of plants. For a specific soil type, ≥ 40 % of total variation in bacterial community ordination could be explained by the molecular composition of SOM. The molecular composition of SOM is proposed as an important factor influencing the microbial response to organic amendments, including biochar.