The effects of biochar application to calcareous soils are not well documented. In a laboratory incubation study, a hardwood-based, fast pyrolysis biochar was applied (0, 1, 2, and 10% by weight) to a calcareous soil. Changes in soil chemistry, water content, microbial respiration, and microbial community structure were monitored over a 12-month period. Increasing biochar application rate increased the water holding capacity of the soil-biochar blend, a trait that could be beneficial under water limited situations. Biochar application also caused an increase in plant-available iron and manganese, soil carbon content, soil respiration rates, bacterial populations, and a decrease in soil nitrate-nitrogen concentration. Biochar rates of 2 and 10% altered the relative proportions of bacterial and fungal fatty acids, and shifted the microbial community towards greater relative amounts of bacteria and less fungi. The ratio of fatty acid 19:0 cy to its precursor, 18:1'7c, was higher in 10% biochar rate soil than all other soils, potentially indicating an environmental stress response. The 10% application rate of this particular biochar was extreme, causing the greatest change in microbial community structure, a physiological response to stress in Gram-negative bacteria, and a drastic reduction in soil nitate-nitrogen (85-97% reduction compared to the control), all of which were sustained over time.