Land use choices differentially affect soil physical and biological properties. Tillage choices in particular affect soil erosion, the retention of soil organic matter, and the biological activity that organic matter supports. The present study evaluated the consequences of different cropping and tillage systems (undisturbed forest, coffee plantation, conventional, and no-tillage row cropping) for soil microbial indicators and sulfur mineralization after 24 years of cropping on an Oxisol (Typic Haplorthox) in an experimental area at Londrina, Brazil. Soil samples were taken at 0–5, 5–10, and 10–20 cm depths and evaluated for microbial biomass P and S, S mineralization, and phosphatase and arylsulfatase activities. Land use affected microbial biomass P and S, and enzyme activity at all depths studied. The cultivated sites had lower values of microbial activity than the undisturbed forested site. Although the coffee site was not tilled and had high organic carbon content, there was low microbial activity, probably due to higher soil acidity and Al content. The estimates of pool stock for microbial P and annual P flux through the soil microbial biomass suggest that these pools are large enough to significantly affect plant nutrient availability. The greater microbial biomass and activity under forested and no-tillage sites may be attributed, at least partially, to higher organic matter content. The soil microbial variables examined proved to be strong indicators of soil sustainability.