Biochar applied to the soil has contributed to the improvement of soil quality, the availability of nutrients for plants, and carbon retention. Besides, the presence of biochar can affect microbial activity. However, information about the action of biochar in the microbial community is scarce. Thus, we hypothesize that biochar, with or without previous addition of NPK fertilizer may alter the activity of some enzymes and total microbial quality of soil. The objective of this study was to evaluate the enzymatic activities and the total microbiological activity of soil amendments with different doses of biochar and grown with sugarcane over time. For two consecutive years, soil samples were collected in the sugarcane planted line amendment with biochar. Sugarcane was planted in 2017, in a randomized block design, a 5 × 2 factorial scheme, with four replications: five doses of biochar (0, 10, 20, 30 and 40 Mg ha−1 (megagram per hectare)), with and without correction of soil fertility. The activities of the enzymes β-glucosidase, acid phosphatase, arylsulfatase, urease, and total microbial quality of the soil were evaluated (fluorescein diacetate method). The application of biochar from eucalyptus residues up to the dose of 30 Mg ha−1 increased the activity of the enzymes β-glucosidase, acid phosphatase, arylsulfatase, urease and the total microbial quality of the soil; however, higher doses reduced the activity of these enzymes and the total microbial quality of the soil over time. The application of mineral fertilizers, in combination with biochar, increased the enzymatic activity of β-glucosidase, arylsulfatase and urease and the total microbial quality of the soil in the soil over time. On the other hand, the addition of NPK fertilizer reduced the activities of the acid phosphatase. •The 30 Mg ha−1 biochar dose in soil resulted in the highest microbiological activity.•Biochar plus NPK increases B-glucosidase and urease activity over time.•Biochar plus NPK in soil increases B-glucosidase, arylsulfatase, and urease activity.•Biochar plus NPK in soil reduces acid phosphatase activity.