Biochar application is a promising strategy for sequestering carbon in agricultural soils and for improving degraded soils. Nonetheless, contradictory and unsettled issues remain. This study investigates whether biochar influences the soil microbial biomass and community structure using phospholipid fatty acid (PLFA) analysis. We monitored the effects of four different types of biochar on the soil microbial communities in three temperate soils of Austria over several months. A greenhouse experiment and two field experiments were conducted. The biochar application did not significantly increase or decrease the microbial biomass. Only the addition of vineyard pruning biochar pyrolysed at 400°C caused microbial biomass to increase in the greenhouse experiment. The biochar treatments however caused shifts in microbial communities (visualized by principal component analysis). We concluded that the shifts in the microbial community structure are an indirect rather than a direct effect and depend on soil conditions and nutrient status.