We examined the effect of wood ash (WA) fertilization on microbial processes in the mineral top soil (0–5 cm) of an acidic Norway spruce forest. Plots treated with a single application of WA (8 t ha⁻¹) were compared with untreated control plots. At five sampling times (1, 4, 15, 62 and 460 d) after the addition of WA, various soil microbiological variables such as microbial biomass C (C_mic), basal respiration (soil native CO₂ release), in situ soil respiration (CO₂ efflux at the soil surface), as well as enzyme activities (acid and alkaline phosphatase, urease, and protease) were determined. Soil chemical properties measured included pH, organic C and total N, and exchangeable cations. WA amendment resulted in a rapid change in the rate of CO₂ evolution and C_mic. It appeared that an increase in microbial activity in soil treated with WA was accompanied by an increase in the growth rate of soil microorganisms. At sampling dates 4 and 15 d after WA application, C_mic and CO₂ evolution rates increased by factors of 1.6 and 1.9, respectively. After 460 d, differences in the CO₂ evolution and C_mic in the control and WA treated plots were still evident. A similar effect could also be shown in situ with higher CO₂ efflux from the soil surface in the WA treated plots. Higher biomass and bioactivity was related to the increases in the pH and in the quantity of nutrients after the WA had been added to the soil. The higher activity was linked to increased mineralization of organic matter, which was followed by decreases in the soil total C and N contents in the first day after treatment. The enzyme activities responded only weakly to the WA treatment. The higher supply of inorganic P from WA led to a small depression in phosphatase activity. In contrast, the enzyme activities of the N-cycle were stimulated by the addition of WA, which had a low N content.