Enzyme activities (EAs) and the FERTBIO sample concept have been increasingly adopted as a novel approach to estimate the soil quality in Brazil. However, the performance of this strategy in sandy soils of the Cerrado biome remains unclear. During 2 years, in a Cerrado's sandy soil, the short-term effects of ten different cropping systems (conventional tillage or no-tillage associated with monoculture, rotations, and/or successions) on the activities of β-glucosidase, acid phosphatase, and arylsulfatase were studied. Issues related to annual variability and the feasibility of using the FERTBIO sample concept for soil enzymes activities were also evaluated. Soil samples were collected at three different depths (0-10 cm, 10-20 cm, and 20-40 cm) in March 2017 and February 2018. Five years since the beginning of the experiment, the presence of cover crops and no-till promoted improvements in EAs evidencing the importance of regenerative management practices for the sustainability of agroecosystems in sandy soils. Regardless of the cropping systems and depths evaluated, soil organic carbon and EAs showed low temporal variation during the 2 years of monitoring. Our results also showed that it is possible to use the FERTBIO sample concept for the Quartzipsament soils of Western Bahia, Brazil.