Fast pyrolysis bio-oil (FPBO) is a liquid biofuel obtained from lignocellulosic residues. Moreover, biomass fly ashes (FAs) containing many minerals and micronutrients are obtained in the production process. Biomass ashes can be used as a lime substitute for amelioration of acid soils by increasing pH, providing nutrients for crop development and stimulating microbial activity. However, ash application might increase N-mineralization and induce nitrate losses via leaching. The main objective of this study was to investigate the applicability of FPBO-recovered FAs as soil amendment and their effects on soil microbial processes, plant development, and to evaluate the effects on soil leaching. In a greenhouse experiment, an acidic soil was amended with 2% of FAs and sown with a regional wheat variety. After 100 days, wheat was harvested and red clover was sown to simulate crop rotation. After 250 days, the soils were analysed microbiologically and physico-chemically. While no differences in plant yields were observed, FAs addition increased several soil chemical pools as well as certain microbiological parameters. Soil pH increased from 4.8 to 7.2, electrical conductivity from 89 to 407 µS cm−1, and the soil available P pool from 13.6 to 81.3 µg g−1 soil. Further, the nitrification rate, nitrate content in the soil leachates increased upon ash addition, in particular during the clover stage of the experiment. Summarized, despite not measurable effects on the plant growth, fly ash appears to enhance chemical and biological properties of soil cropped with wheat and clover without hinting towards negative environmental side-effects. Ministerio de Economía, Industria y Competitividad | Ref. RYC-2016–21231