Microorganisms respiratory and enzymatic activities provide sensitive indicators of changes in soil properties, such as those caused by interactive effects of tillage and fertilization regimes or other agricultural practices. However, the rapid, adaptive microbial growth, respiratory and enzymatic responses to changes in soil environments induced by specific agricultural practices are not well understood. Thus, to explore these adaptations we compared effects of contrasting environments on functional microbial traits (growth and enzyme kinetic parameters) in a Mediterranean agro-ecosystem. These environments differed in long-term disturbance (no, minimum, or conventional tillage), nitrogen-richness (fertilization with 90 kg N ha−1 versus no fertilization), and resource scarcity (increasing with soil depth in 0–30, 30–60 and 60–90 cm layers). Reducing soil disturbance from conventional to minimum tillage promoted microbial growth through shorter Tlag and larger active biomass fraction and induced increases in N- and P-acquiring enzyme activities by increasing nutrients limitation. Fertilization stimulated increases in fast-growing microorganisms with low substrate-affinity enzyme systems, microbial biomass, enzymatic activities, and turnover rates of soil organics. In contrast, increasing scarcity of resources with soil depth strongly reduced microbial biomass and activity. A lack of correlation between soil and enzymatic stoichiometric ratios raises concern regarding the applicability of eco-enzymatic stoichiometric indexes in Mediterranean agro-ecosystems. We conclude that decomposition and turnover of organic substrates under contrasting agricultural practices are mediated by microbial communities with distinct functional traits (active fraction, growth parameters) and enzyme properties (Vmax, Km), which need to be considered in smart land use regimes.