Healthy soils are the second C sink on Earth, and this sink could last for hundreds or even thousands of years as stable soil organic matter (SOM). Forest soils, in particular, have the potential to store significant amounts of C, however, the amount of C sequestered and the carbon-to-nitrogen (C/N) ratio of soil organic matter (SOM) depend on the vegetation influencing the soil. In the last decades, mixed stands have aroused great interest among the scientific community, but it is still necessary to intensify research on its effect on soils and their C storage capacity. In this study, we assess soil C sequestration potential in soil and litter of mixed and pure stands of Scots pine and beech (Pinus sylvestris–Fagus sylvatica). Three triplets (9 forest plots), two located in Southern Poland and one in Southern Germany were studied. A total of 40 circular subplots of 5 m radius were selected within the triplets, covering a wide range of species mixture, and soil and litter were sampled. Data were analyzed at two scales (plot-level and microsite-level) to determine which option is more appropriate when studying the mixing effect on SOM. Cstock in forest floor ranged between 2.5 and 11.1 Mg C ha−1 and in mineral soil between 39.6 and 337.8 Mg ha−1. According to our findings, the percentage of species mixture primarily impacted the forest floor rather than the mineral soil. On the forest floor, stands with 25–50% pine in the mixture were found to have a C/N ratio between 20 and 30, which indicates an equilibrium state between mineralization and immobilization. In the mineral soil, total organic C was the only variable affected by mixture percentage (p < 0.1). Finally, microsite-level scale proved to be the most appropriate when studying tree stand composition effect on SOM, as the plot-level scale diluted or masked some effects.