In the south of Brazil, the intensification of eucalyptus planting can impact the local biome, characterized by grasslands that extends to Uruguay and Argentina. Thus, the objective of the study was to evaluate changes in soil carbon, nitrogen and their labile fractions and soil microbiological attributes due to the introduction of eucalyptus cultivation in the Pampa biome. The soils evaluated were Eutric Regosol (RG), Haplic Lixisol (LX), Dystric Leptosol (LP) and Pellic Vertisol (VR). For this, areas of homogeneous cultivation of Eucalyptus saligna (7 years old) and areas of grassland (native vegetation) were selected. Physical granulometric fractionation of soil organic matter (SOM) was performed in the 0–5, 5–10 and 10–15 cm soil layers, quantifying levels, stocks and distribution of total organic carbon (TOC) and total nitrogen (TN) in the labile and associated with minerals fractions, and the carbon management index (CMI) was calculated. In the 0–5 and 5–10 cm soil layers, microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), basal soil respiration, metabolic coefficient, MBC / TOC, MBN / TN, MBC / MBN ratios were also evaluated. Eucalyptus cultivation increased the TOC and POC carbon stocks by 8.7 and 21.7%, respectively (0-15 cm) in compared to grassland. It was also observed that the cultivation of eucalyptus increased 6.7% and 26.0% the stocks of TN and PN, respectively, compared to grassland in the pampa biome. Carbon stocks associated with minerals were not affected by eucalyptus cultivation. The cultivation of eucalyptus increased the basal respiration and the microbial biomass carbon in relation to grassland and there is a reduction of microbial biomass nitrogen in the soil. Thus, the cultivation of eucalyptus in the Pampa biome in southern Brazil demonstrates that it is a forest crop that can contribute to the increase in total and labile carbon stocks and also the biomass and microbial activity of the soil, which are important indicators of assessment of soil quality.