Biochars are often proposed as a strategy for long-term carbon sequestration. Nevertheless, application of pyrolysed feedstock, particularly along with exogenous organic matter, may affect carbon dynamics in soil through introduction of labile carbon pools and stimulation of extracellular enzymes activity. The main aim of this research was to evaluate the influence of biochars and unprocessed organic amendments addition in two agricultural soils on the dissolved organic carbon (DOC) content and activity of three enzymes involved in carbon turnover. In the incubation experiment, activity of dehydrogenase, β-glucosidase, cellulase and DOC content were measured on day 30, 60, 90, 180 and 360. Addition of biochars stimulated the activity of dehydrogenase and β-glucosidase, while cellulase was suppressed. Fresh biomass amendment enhanced activity of the enzymes through priming effect. DOC content tended to be the highest in treatments with high enzyme activity, suggesting that DOC introduced with amendments acted as a source of energy for microbes. Our findings support the hypothesis that biochar properties and presence of exogenous organic matter affect microbial response in soil, what might be crucial for carbon sequestration potential of biochar. However, long-term studies are recommended to fully understand the mechanisms that determine response of soil biota to biochar addition.