The ability of the soil's biological community to immobilise carbon (C) from substrates, often referred to as carbon use efficiency (CUE), has been shown to be dependent on the prevailing soil conditions and management regime, potentially leading to changes in C storage and functioning. However, there remains a lack of understanding about how soil CUE is affected by different common labile substrates (and combinations thereof) and native soil organic matter (SOM) status. Here we studied the CUE of three ubiquitous soil C substrates central to microbial metabolism, namely glucose, glutamic acid and citric acid, in soils with and without long-term C deprivation and associated differences in microbial biomass and community structure. We hypothesised that C deprivation-induced stress would reduce substrate CUE due to the investment of more C into stress-alleviation metabolic pathways, while conversely a balanced mixture of C substrates (i.e., sugars, amino acids and organic acids) would promote more efficient growth and substrate CUE. Our results showed that CUE was substrate-specific following the series glucose > glutamic acid > citric acid. CUE values for glucose demonstrated plasticity, being significantly lower in the soils experiencing C deprivation for both 6 and 16 years. Further, the CUE of glucose was increased when supplied alongside citrate and glutamate suggesting that substrate mixture may promote more efficient microbial growth. In contrast, the CUE for glutamic acid and citric acid showed no plasticity, being unaffected by both SOM status and the presence of other substrates. In conclusion, we found evidence to support both our hypotheses indicating that the type of C entering soil alongside native SOM levels may have a strong influence on overall CUE and thus C storage potential.