The Atlantic Forest exhibits remarkable floristic diversity over short distances, and when combined with altitude, it results in variations in soil biological properties and hydrological dynamics. This study aimed to quantify the distribution pattern of soil biological properties along a small topographic gradient and identify how these properties are related to soil chemical, physical, and topographical attributes in an Atlantic Forest fragment in Brazil. Plots were established along hillslope positions varying from 100 to 180 m of altitude. Soil biological characterization was performed at a depth of 0.0–0.10 m in both summer and winter seasons. The lowland showed higher microbial biomass nitrogen in summer (57.28 ± 5.57 μg g−1 soil) and higher anaerobically mineralizable nitrogen in summer and winter (42.70 ± 2.14 mg kg−1 and 41.06 ± 3.37 mg kg−1, respectively). The midland showed lower microbial biomass carbon, enzymatic activity, and soil moisture in both seasons, and higher metabolic coefficients in summer. Soil chemical properties exerted a greater influence on the variability of biological properties in both seasons. The land slope conditioned lower microbial activity and organic cycling in the midland. Soil biological properties were affected by seasonality. Even a small altitudinal gradient (up to 100 m) in the Sea of Hills regions of the Atlantic Forest can lead to significant changes in soil biological and chemical attributes.