Background and aims: Biochar is produced from the pyrolysis of organic materials, and when buried in soil can act as a long term soil carbon (C) store. Evidence suggests that biochar can also increase crop yields, reduce nutrient leaching and increase biological nitrogen fixation in leguminous plants. However, the potential for increasing biological N2 fixation in agroecosystems is poorly understood, with inconsistent reports of root nodulation following biochar application. Therefore, the aim of this study was to determine the effect of biochar application rate and time since application on nodulation and nitrogenase activity in nodules of clover grown in a temperate agricultural soil. Methods: We used replicated field plots with three biochar application rates (0, 25 and 50 t ha-1). Three years after biochar amendment, the plots were further split and fresh biochar added at two different rates (25 and 50 t ha-1) resulting in double-loaded reapplications of 25 + 25 and 50 + 50 t ha-1. Results: Three years after biochar application, there was no significant difference in the total number of root nodules between biochar-amended and unamended soil, regardless of the application rate. However, despite clover root nodules being of a similar number and size the level of nitrogenase activity of individual nodules in biochar-amended soil was significantly higher than in unamended soil. Reapplication of biochar resulted in decreased nodulation, although the rate of nitrogenase activity per nodule remained unaffected. Conclusion: In the short term, biochar influences root nodule number and localised N2 fixation per nodule; however, total nitrogenase activity for the whole root system remained unaffected by the application rate of biochar or time since its application. These results emphasise the importance of long-term field studies, with a variety of applications rates for determining the influence of biochar applications on N2-fixing organisms and in providing data that can meaningfully inform agronomic management decisions and climate change mitigation strategies.