Conservation agriculture (CA) is an essential strategy to avoid agricultural land degradation. It affects nutrient availability to plants and soil biological activity. However, an integral view of the effects of CA on soil fertility and biological indicators has been rarely described. Here, we examined the long‐term effects of no‐till and residue mulching on chemical properties, biological functionality indicators (enzyme activity), and nutrient availability indices of soil, and also their potential relationships, in order to provide a comprehensive view of the effects of CA on soil quality improvement. To this end, we assessed the effects after 27 years of conventional tillage (CT) and no‐tillage (NT) on a vertisol. NT increased Fe and Mn bound to organic matter when compared with CT. P and K availability in the soil surface was greater under NT than under CT by effect of more marked fertilizer stratification, and in the case of P by a decreased precipitation rate of Ca phosphates ascribed to an increased soil organic matter (SOM). The availability indices for Fe, Mn, Cu, P, and B were correlated with some enzyme activities. Our results reveal a relationship of soil biological functionality indicators, which were higher under CA, to nutrient availability to plants. This was explained at least in part by changes in soil properties, in particular an increased SOM, affecting both biological properties and nutrient availability. Thus, enzyme activities can be considered effective indicators of change in soil fertility, that is, nutrient availability to plants, a crucial factor for soil quality and functioning.