Biological parameters are used as early indicators of soil quality (SQI) due to their quick response to anthropic management. The objective of this research was to evaluate the effect of tillage system (NT – no tillage and CT- conventional tillage), nitrogen fertilization (N0, N1, N2 and N3), and crop rotation (bean-corn and bean-amaranth), on physical, chemical, and biological quality indicators of an agricultural soil. The study was carried out on a Mollisol. Evaluated indicators were soil moisture,
soil stable aggregates, pH, particulate organic matter, C: N ratio, organic carbon, microbial biomass, microbial respiration, enzymatic activity [acid phosphatase, Fluorecin Di acetate (FDA) hydrolysis activity, β-glucosidase (β-G) and total glomalin content (TG)]. The results showed that acid phosphatase had the highest activity under CT with the bean-corn rotation; the FDA responded to the effect of the bean-corn rotation under high N levels; the β-G enzyme activity was higher under CT with both rotations; finally, TG responded to NT management with the bean-corn rotation. Also, the principal component analysis allowed to infer that all evaluated biological characteristics are SQI. It is concluded that the soil biological indicators evaluated were affected by the studied factors.