AbstractFungi and bacteria are the two principal microbial groups in soil, responsible for the breakdown of organic matter (OM). The relative contribution of fungi and bacteria to decomposition is thought to impact biogeochemical cycling at the ecosystem scale, whereby bacterially dominated decomposition supports the fast turnover of easily available substrates, whereas fungal‐dominated decomposition leads to the slower turnover of more complex OM. However, empirical support for this is lacking. We used soils from a detritus input and removal treatment experiment in an old‐growth coniferous forest, where above‐ and belowground litter inputs have been manipulated for 20 yr. These manipulations have generated variation in OM quality, as defined by energetic content and proxied as respiration per g soil organic matter (SOM) and the δ13C signature in respired CO2and microbial PLFAs. Respiration per g SOM reflects the availability and lability of C substrate to microorganisms, and the δ13C signature indicates whether the C used by microorganisms is plant derived and higher quality (more δ13C depleted) or more microbially processed and lower quality (more δ13C enriched). Surprisingly, higher quality C did not disproportionately benefit bacterial decomposers. Both fungal and bacterial growth increased with C quality, with no systematic change in the fungal‐to‐bacterial growth ratio, reflecting the relative contribution of fungi and bacteria to decomposition. There was also no difference in the quality of C targeted by bacterial and fungal decomposers either for catabolism or anabolism. Interestingly, respired CO2was more δ13C enriched than soil C, suggesting preferential use of more microbially processed C, despite its lower quality. Gross N mineralization and consumption were also unaffected by differences in the ratio of fungal‐to‐bacterial growth. However, the ratio of C to gross N mineralization was lower than the average C/N of SOM, meaning that microorganisms specifically targeted N‐rich components of OM, indicative of selective microbial N‐mining. Consistent with the δ13C data, this reinforces evidence for the use of more microbially processed OM with a lower C/N ratio, rather than plant‐derived OM. These results challenge the widely held assumption that microorganisms favor high‐quality C sources and suggest that there is a trade‐off in OM use that may be related to the growth‐limiting factor for microorganisms in the ecosystem.