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ABSTRACT There are few long-term field studies on the effects of biochar on soil microbial abundance and diversity. This study aimed to evaluate doses of biochar in combination with mineral fertilizer on the activity and diversity of microorganisms in the soil of a sugarcane field. The experiment was carried out in a randomized block design, factorial 5 × 2, with four replications: five doses of eucalyptus (Eucalyptus grandis Hill ex Maiden × Eucalyptus urophylla S.T. Blake) wood biochar (0, 10, 20, 30, and 40 Mg ha−1), with and without the application of nitrogen, phosphorus, and potassium (NPK) mineral fertilizer. Soil samples were collected from the sugarcane planting line and fertilized with biochar for two consecutive years. Regardless of the NPK fertilizer, over the two years of evaluation, the height growth of sugarcane plants and total organic carbon (TOC) increased linearly with biochar doses. For microbial biomass carbon (Cmic), soil basal respiration (SBR), metabolic quotient (qCO2), microbial quotient (qMIC), and Shannon diversity index (H), the highest values were obtained where fertility correction (WFC) treatments were applied, regardless of the year of evaluation, in biochar doses between 20 and 30 Mg ha−1. On the other hand, the highest CO2 efflux values were obtained with zero doses of biochar, regardless of the NPK fertilizer applied, over the two years of evaluation. Therefore, the incorporation of biochar and NPK fertilizer into the soil contributes to increasing the soil's biological activity indicators and, consequently, the growth of sugarcane plants. It is essential to highlight the need for continuous assessments as the characteristics of biochar change over time. longterm long term design 2 replications ST S T S.T Blake 0, 0 (0 10 4 ha−1, ha1 ha ha−1 , 1 ha−1) nitrogen phosphorus (NPK evaluation TOC (TOC Cmic, Cmic (Cmic) SBR, SBR (SBR) qCO2, qCO2 qCO (qCO2) qMIC, qMIC (qMIC) H, H (H) WFC (WFC applied 3 hand CO Therefore soils s consequently time ( ha− (Cmic (SBR (qCO2 (qMIC (H (qCO