Soil microbial growth and respiration play a critical role for soil organic carbon dynamics. Yet, we lack understanding of the main controls of soil microbial carbon metabolism at large scales. Here, we investigated whether and how the chemical composition of microbially available organic matter affects soil microbial carbon metabolism across soil systems. We linked soil microbial growth and respiration rates as well as carbon use efficiency (quantified with 18O stable isotope probing) to the chemical composition of extractable organic matter (characterized with reversed-phase liquid chromatography coupled to Fourier-transform ion cyclotron resonance mass spectrometry) along a geoclimatic gradient of 33 Chilean temperate grassland soils. We found that biomass-normalized rates of growth and respiration were primarily positively linked to aliphatics such as carbohydrate-, proteinaceous- and amino sugar-like compounds, and secondarily to unsaturated lignin-like compounds. Respiration was positively linked to compounds with carbon in a reduced oxidation state, suggesting carbon-conserving catabolism, while growth appeared unrelated to the oxidation state of carbon. This suggests that other mechanisms than mere energetic constraints control microbial growth rates in aerated soils. Our findings demonstrate that information on the chemical composition of bioavailable organic matter can provide insights into the processes that govern the fate of carbon across different ecosystems.