The interplay between soil microbial anabolism and metabolism has recently been postulated as an important lever for carbon sequestration in soils. Theoretical considerations suggest that high microbial growth rates and a high carbon-use efficiency (CUE) are associated with increased soil organic carbon (SOC) build-up in arable soils. Nature-oriented conservation agricultural practices are believed to facilitate this microbial-driven process of SOC accrual; a practical validation of this postulation is however missing. Against this background, we evaluated the effect of agricultural management and site-specific soil chemical and physical properties on microbial physiology (i.e., respiration, bacterial and fungal growth rates, and microbial biomass turnover) and CUE by an on-farm comparison of conventional farming, conservation farming and unmanaged reference soil systems (i.e., adjacent field margins) at twenty-one sites across three soil depths. We show that a change in agricultural management towards soil health regeneration has a significant impact on microbial physiology. Bacterial growth, respiration and microbial biomass turnover increased from conventional farming towards conservation farming systems to reference soils. The opposite trend however was found for fungal growth and microbial CUE, with highest values observed in conventional farming systems. This clearly contradicts the prevailing idea that higher microbial CUE in conservation farming systems may be associated with higher SOC contents. Our results further show that soil physical and chemical parameters such as soil pH, texture and dissolved carbon compounds strongly predict microbial CUE. In line with these results, functional pore domains showed specific optima for microbial growth and CUE. For example, a high share of small pore domains hampered fungal growth and reduced microbial CUE but facilitated microbial biomass turnover at the same time. Thus, we suggest that – superimposed upon agricultural management effects – habitat structural conditions and microbial carbon limitation mainly shape microbial physiology and CUE in arable soil systems.