Wood ash application (WAA) to the forest has been suggested as a resource of nutrients for trees and for restoration of acidified soils and surface waters. However, studies of the effects of WAA on aquatic systems are few. This study investigated the effects of wood ash (1–25 g L⁻¹) on the unicellular flagellate Euglena gracilis, which has been proved to be a sensitive test organism for assessing environmental change. Long-term (7 days) growth studies and short-term (direct, 24 and 48 h) studies of different motile factors, using the automatic biological test system Ecotox, were conducted. The results show no indication of biotoxic effects due to wood ash (adjusted to neutral) treatments. However, when no adjustments of pH in wood ash solutions were made, inhibitory effects on motile factors and cell growth were observed at higher concentrations of wood ash (10–25 g L⁻¹) due to high pH (> 8). In tests with pH adjusted to neutral, enhanced motility was observed. These results indicate that high concentration of WAA could affect freshwater environments.