Soil CO2 efflux is a complex flux composed of autotrophic and heterotrophic sources of the CO2. To correctly estimate whether soil is and will be acting as a sink or a source for atmospheric CO2, these two principal components should be studied separately. This is because they respond differently to biological and environmental drivers. Studies which compare partitioning methods of soil CO2 in one single study using unique measurement equipment are lacking instead. In this study we attempted to compare three partitioning methods of soil CO2 efflux: mesh exclusion, combined SIR and regression method and to choose the most appropriate for herbaceous ecosystems (e.g. grasslands and urban lawns). Mesh exclusion and combined SIR demonstrated comparable results in determination of autotrophic and heterotrophic contribution to CO2 efflux and the methods were defined as suitable. Regression method necessitates ulterior improvements prior application in herbaceous ecosystems. © Springer International Publishing AG, part of Springer Nature 2019.