The choice of cultivation system in arable agriculture exerts a strong influence not only on soil health and crop productivity but also on the wider environment. Conservation tillage using non-inversion methods conserves soil carbon, reduces erosion risk and enhances soil quality. In addition, conservation tillage has been shown to sequester more carbon within the soil than inversion tillage, reducing carbon dioxide losses to the atmosphere. Stable, well structured topsoils that develop following long-term conservation tillage lead to more energy efficient systems due to the reduced power requirements for cultivation. Long-term experiments, e.g. more than 20 years, that confirm the impact of conservation tillage over an extended period are not common. Here we evaluate the impact of different tillage methods and winter wheat straw management, either incorporated or removed, on organic matter turnover and soil quality indicators. No-till, chisel and mouldboard ploughing was carried out for 23 years on a silty clay loam soil in South West England that was not considered suitable for non-inversion tillage due to weak soil structure. In order to assess the effect of contrasting cultivation and straw disposal method on soil carbon dynamics, a range of assays were conducted, including water extractable organic carbon, hot water extractable carbohydrate, microbial biomass carbon, activity of beta-glucosidase and acid phosphatase enzymes, C sequestration and the natural abundance of 13C. Our results show that the soil organic carbon concentration in the topsoil was greater under no-till than mouldboard ploughing, while a reverse trend was observed in the lower depths. A 14–17% increase in soil organic carbon was observed in the topsoil for chisel plough and no-till treatments compared to mouldboard ploughing. Water extractable organic carbon was found to constitute only 1–7% of the microbial biomass carbon. Hot water extractable carbohydrate was one of the most sensitive indicators of soil quality and had a significant a negative correlation with bulk density and positive correlation with soil organic carbon microbial biomass carbon beta-glucosidase and acid phosphatase. The choice of cultivation method exerted a major control on microbial and carbon dynamics. No-till and chisel ploughing maintained carbon in the soil surface horizons, which will benefit the stability of this weakly structured soil, but mouldboard ploughing distributed carbon more uniformly throughout the soil profile, particularly when straw was incorporated, hence leading to the retention of more carbon in the soil profile.