The management of crop residues by tillage is a key determinant that affects microbial processes, leading to changes in soil organic carbon (SOC) pools and fertility status. However, studies evaluating the long-term temporal changes caused by no-tillage (NT) system are still incipient. Therefore, the objective of this study was to evaluate the effects of a 22-year tillage chronosequence on microbial activity and soil fertility attributes in a subtropical ecosystem in Brazil. Field experiments were conducted at two research sites. One site was located near Tibagi (Santa Branca Farm), and the other site was located near Ponta Grossa City (Frankanna Farm), Paraná State, Brazil. The soils are classified as Typic Hapludoxes and the tillage chronosequence comprised five treatments: (i) native vegetation (NV); (ii) NT for 10 years (NT-10); (iii) NT for 20 years (NT-20); (iv) NT for 22 years (NT-22); and (v) conventional tillage for 22 years (CT-22). We determined soil microbial biomass carbon (MB-C) and nitrogen (MB-N), basal respiration (BR), microbial quotient (MB-C and SOC ratio), metabolic quotient (qCO2), total polysaccharides (TP), and fertility attributes (pH, N, S, P and effective CEC). A strong interaction between soil management and microbial activity soil fertility attributes was observed. NV exhibited higher MB-C and MB-N and lower BR and qCO2, suggesting that NV could support a more stable ecosystem. CT-22 depleted soil C, N, S and P concentrations, but increasing NT time restored their concentrations. There were 34%, 42% and 48% less SOC (-14.9 g C kg-1), N (-1.4 g N kg-1) and S (-0.13 g S kg-1), respectively, in CT-22 in relation to the NT-22 soil at 0 to 5 cm depth. In addition, CT-22 showed significantly (p¿<¿0.05) lower MB-C, MB-N and a high qCO2, indicating intense competition among soil microbes for available C. The study emphasized NT system potential to sequester C in soils. Although further studies are necessary to confirm this hypothesis, it can ind