Biochar as a carbon-rich highly porous substance has been proposed for use in agriculture and horticulture as a soil amendment. One of the main concerns of this application of biochar is its potential contamination with heavy metals (HMs) and polycyclic aromatic hydrocarbons. The aim of this research was to access the environmental risk of biochar used as a soil amendment on soil mesofauna (mites and springtails). We conducted both field and laboratory experiments with the use of wood-chip biochar from low-temperature (300 °C) flash pyrolysis. Biochar was free from polycyclic aromatic hydrocarbons (PAH), and the concentration of all tested toxic compounds was very low or even under the level of detection. Both the results of field and laboratory studies show no toxic effects on soil mesofauna. In the field studies, the biochar application of 50 t/ha in maize and oilseed rape crops significantly increased the mean number of mesofauna. This change probably resulted from improved soil chemical properties (in particular organic carbon content and cation exchange capacity) upon biochar addition. The results of the avoidance test with the use of springtail species Folsomia candida showed the possible short-term toxicity risk from a dose of 5%. The results of the reproduction test indicate the negative response of F. candida from the rate of 25% (higher than the field dose, which corresponds to 10% in laboratory tests). The reason for the short-term toxicity might be the considerable increase in soil pH after biochar addition. To our knowledge, this is the first study that has looked so widely into the effect of biochar on soil mesofauna. We encourage further studies into the risk assessment of biochar on soil organisms in both a controlled laboratory environment and in the open field.