The production of fast pyrolysis bio oil (FPBO) constitutes one of the newest technologies for gaining a liquid biofuel from woody biomass. During this process biomass fly ashes (FAs), rich in minerals and salts, are produced. However, FAs are often disposed in landfills and their fertilising potential has been underestimated. A greenhouse trial was set up to test the impact of FA on soil physico-chemical and microbiological properties with a special focus on phosphorus, one of the main limiting nutrients in terrestrial ecosystems. FA were added into an acidic grassland soil at a rate of 2% with wheat (Triticum aestivum subsp. spelta) used as test plant. Soil and plants were collected after an incubation period of 60 and 100 days. Ash application increased soil pH and electrical conductivity, and improved soil nutritional status by increasing soil total, inorganic, and plant available phosphorus over time. Accordingly, higher plant yields were observed in ash-treated soils. The effect of FA on microbial biomass, assessed as double stranded DNA content, was time dependent and increased significantly with plant presence. Acid phosphomonoesterase activity significantly decreased following ash addition. However, neither alkaline phosphomonoesterase (ALP) activity nor the abundance and composition of the ALP gene (phoD) harboured by bacteria were affected by FA application. On the whole, FA from FPBO production seems to improve soil nutrient status and plant growth without inheriting detrimental effects on soil microbial communities in the mid-term.