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At present, there is limited understanding of how biochar application to soil could be beneficial to crop growth in temperate regions and which biochar types are most suitable. Biochar’s (two feedstocks: willow, pine; three pyrolysis temperatures: 450 °C, 550 °C, 650 °C) effect on nitrogen (N) availability, N use efficiency and crop yield was studied in northwestern European soils using a combined approach of process-based and agronomic experiments. Biochar labile carbon (C) fractions were determined and a phytotoxicity test, sorption experiment, N incubation experiment and two pot trials were conducted. Generally, biochar caused decreased soil NO3− availability and N use efficiency, and reduced biomass yields compared to a control soil. Soil NO3− concentrations were more reduced in the willow compared to the pine biochar treatments and the reduction increased with increasing pyrolysis temperatures, which was also reflected in the biomass yields. Woody biochar types can cause short-term reductions in biomass production due to reduced N availability. This effect is biochar feedstock and pyrolysis temperature dependent. Reduced mineral N availability was not caused by labile biochar C nor electrostatic NH4+/NO3− sorption. Hence, the addition of fresh biochar might in some cases require increased fertilizer N application to avoid short-term crop growth retardation.