Using O₂ stable isotope analysis, we determined the isotopic fractionation of biological O₂ consumption by 10 flavin-dependent enzymes and 6 metalloenzymes. Metalloenzymes displayed a narrower range and lower values of isotopic fractionation than flavin-dependent enzymes. This work expands our understanding of the variability of oxygen isotopic fractionation at the enzyme level, improving the ability to study O₂ dynamics from molecular to ecosystem scales.