Water is one of the main variables affecting the carbon cycle (carbon storage or assimilation) in soils or litters from the Mediterranean area, though in most studies it has been considered via soil moisture measurements only. Here, we investigated the effect of water availability as characterized by water activity, aw, on the equilibrium of enzyme reactions (hydrolysis/synthesis) for litters from three species characteristic of the Mediterranean area (Quercus pubescens L, Q. ilex L. and Pinus halepensis L.). Lipases were used as models and an organic phase was used as the reaction medium to adjust water amount. We found that the activities of hydrolysis/transesterification increased with aw, showing that both can occur at the same aw in litter. Furthermore, these lipase activities in litter decreased for aw close to 1, which has been described with purified lipases in organic media, indicating potential enzyme aggregation. Variations in aw with moisture were studied with sorption isotherms, which were found to be similar (isotherm type 2) for all the studied litters. Water activity is a crucial indicator for soils under hydric stress at small water contents, which should be considered for describing more precisely enzyme functioning and giving valuable information about carbon dynamics in soils or litters.