Applying crop residues is a widely used strategy to increase soil organic matter (SOM) in arable soils because of its recorded effects on increasing microbial biomass and consequently necromass. However, fresh residue inputs could also prime the decomposition of native SOM, resulting in accelerated SOM depletion and greenhouse gas (GHG) emission. Increasing the botanical diversity of the crops grown in arable systems has been promoted to increase the delivery of multiple ecological functions, including increasing soil microbial biomass and SOM. Whether mixtures of fresh residues from different crops grown in polyculture contribute to soil carbon (C) pools to a greater extent than would be expected from applying individual residues (i.e., the mixture produces a non-additive synergistic effect) has not been systematically tested and is currently unknown. In this study, we used 13C isotope labelled cover crop residues (i.e., buckwheat, clover, radish, and sunflower) to track the fate of plant residue-derived C and C derived from the priming of SOM in treatments comprising a quaternary mixture of the residues and the average effect of the four individual residues one day after residue incorporation in a laboratory microcosm experiment. Our results indicate that, despite all treatments receiving the same amount of plant residue-derived C (1 mg C g-1 soil), the total microbial biomass in the treatment receiving the residue mixture was significantly greater, by 26% (3.69 μg C g-1), than the average microbial biomass observed in treatments receiving the four individual components of the mixture one day after applying crop residues. The greater microbial biomass C in the quaternary mixture, compared to average of the individual residue treatments, that can be attributed directly to the plant residue applied was also significantly greater, by 132% (3.61 μg C g-1). However, there was no evidence that the mixture resulted in any more priming of native SOM than average priming observed in the individual residue treatments. The soil microbial community structure, assessed using phospholipid fatty acid (PLFA) analysis, was significantly (P < 0.001) different in the soil receiving the residue mixture, compared to the average structures of the communities in soil receiving four individual residues. Differences in the biomass of fungi, general bacteria, and Gram-positive bacteria were responsible for the observed synergistic effect of crop residue mixtures on total microbial biomass and residue-derived microbial biomass, especially biomarkers 16:0, 18:2ω6 and 18:3ω3. Our study demonstrates that applying a mixture of crop residues increases soil microbial biomass to a greater extent than would be expected from applying individual residues and that this occurs either due to faster decomposition of the crop residues or greater carbon use efficiency (CUE), rather than priming the decomposition of native SOM. Therefore, growing crop polycultures (e.g., cover crop mixtures) and incorporating mixtures of the resulting crop residues into the soil could be an effective method to increase microbial biomass and ultimately C stocks in arable soils.