There is substantial variation in estimates of the respiratory quotient (RQ), i.e., molar ratio of produced CO₂ and consumed O₂ during microbial mineralization of organic matter (OM). While several studies have examined RQ's controlling factors in terrestrial or aquatic ecosystems, there are no broader cross-ecosystem comparisons, and there is a lack of general understanding of the extrinsic (environmental) and intrinsic (organic matter composition) controls on RQ. In this study, we examine RQ across a broad range of environments, including soils, aquatic sediments, lake and coastal water. We measured CO₂ production and O₂ consumption using membrane inlet mass spectrometry (MIMS). We also assessed the microbial metabolic profiles using BIOLOG EcoPlates and determined the energy content of the natural OM with bomb calorimetry and its elemental composition. We show that RQ differs significantly between the ecosystem types and strongly deviates from the frequently assumed value of 1. In addition, microbial mineralization across the different studied ecosystems is correlated with the bulk energy content of the OM (kJ g^-1 organic carbon). Finally, RQ was correlated to the metabolic profiles of microorganisms, as estimated based on BIOLOG EcoPlates. We argue that an increased use of cross-ecosystem experimental studies will enhance the understanding of the factors controlling carbon cycling.