Four biochar types, produced by slow pyrolysis of poultry litter (PL) and pine chips (P) at 400 or 500 °C, were added to two adjacent soils with contrasting soil organic matter (SOM) content (8.9 vs. 16.1 g C kg−1). The N mineralization rate was determined during 14-week incubations and assessments were made of the microbial biomass C, dehydrogenase activity, and the microbial community structure (PLFA-extraction). The addition of PL biochars increased the net N mineralization (i.e., compared to the control treatment) in both soils, while for treatments with P biochars net N immobilization was observed in both soils. Increasing the pyrolysis temperature of both feedstock types led to a decrease in net N mineralization. The ratio of Bacterial to Fungal PLFA biomarkers also increased with addition of biochars, and particularly in the case of the 500 °C biochars. Next to feedstock type and pyrolysis temperature, SOM content clearly affected the assessed soil biological parameters, viz. net N mineralization or immobilization, MBC and dehydrogenase activity were all greater in the H soil. This might be explained by an increased chance of physical contact between the microbial community activated by SOM mineralization upon incubation and discrete biochar particles. However, when considering the H soil's double C and N content, these responses were disproportionally small, which may be partly due to the L soil's, somewhat more labile SOM. Nonetheless, increasing SOM content and microbial biomass and activity generally appears to result in greater mineralization of biochar. Additionally, higher N mineralization after PL addition to the H soil with lower pH than the L soil can be due to the liming effect of the PL biochars.