Biochar application to the soil has been recommended as a carbon (C) management approach to sequester C and improve soil quality. Three-year experiments were conducted to investigate the interactive effects of three types of amendments on microbial biomass carbon, soil dehydrogenase activity and soil microbial community abundance in luvisols of arable land in the Czech Republic. Four different treatments were studied, which were, only NPK as a control, NPK + cattle manure, NPK + biochar and NPK + combination of manure with biochar. The results demonstrate that all amendments were effective in increasing the fungal and bacterial biomass, as is evident from the increased values of bacterial and fungal phospholipid fatty acid analysis. The ammonia-oxidizing bacteria population increases with the application of biochar, and it reaches its maximum value when biochar is applied in combination with manure. The overall results suggest that co-application of biochar with manure changes soil properties in favor of increased microbial biomass. It was confirmed that the application of biochar might increase or decrease soil activity, but its addition, along with manure, always promotes microbial abundance and their activity. The obtained results can be used in the planning and execution of the biochar-based soil amendments.