<p>Recent studies suggest that biochar may enhance biological nitrogen fixation (BNF) in legumes, yet the underlying mechanisms remain unclear. This study aimed to explore the potential effects of manure-derived biochar on BNF, with a focus on its influence on soil physicochemical properties and the availability of essential nutrients such as molybdenum (Mo), a cofactor for nitrogenase. A greenhouse experiment was conducted using dairy manure biochar or lime applied to soils growing white clover (<i>Trifolium repens</i>), either with or without phosphorus (P) and potassium (K) fertilizers. Plant growth, nutrient uptake, nitrogenase activity, and post-harvest soil properties were evaluated. Biochar application significantly increased soil pH from 6.0 to nearly 7.0 and the available Mo content by 16–151%. In contrast, lime raised soil pH to a similar extent but did not significantly affect available Mo content, which highlights the role of biochar as a direct Mo source. Biochar also significantly promoted plant growth—particularly root biomass when combined with PK fertilizers—and markedly increased the abundance of nitrogen-fixing bacteria in roots. Biochar-treated plants showed enhanced uptake of Mo along with increased nitrogen content. Although the acetylene reduction assay showed only a slight increase in nitrogenase activity, biochar-treated plants exhibited improved uptake of P, K, and S, all of which support nitrogenase function. These findings suggest that dairy manure biochar promotes BNF by improving soil pH, increasing Mo availability and uptake, stimulating root growth, enhancing nutrient acquisition, and fostering associations with nitrogen-fixing bacteria.</p> Graphical Abstract <p></p>