The aim of this study was to investigate the influence of the water activity on the stability of lyophilised lactic acid bacteria, especially in the solid glassy region. Lactobacillus bulgaricus CFL1 was co-lyophilised with sucrose and stored under controlled relative humidity at 25 °C. Glass transition temperature (T g), water activity, water content and loss of specific acidification activity during storage were determined. The rates of bacteria degradation were analysed as a function of water activity and as a function of the temperature difference between storage temperature and T g. Above T g , the degradation rate appeared related to the physical changes of the amorphous matrix. Below T g , the optimal stability of the lyophilised bacteria was observed in the intermediate water activity range 0.1–0.214. An integrated analysis of the relationships between water activity, T g , water content and biological activity appeared as a promising approach for optimising the freeze-drying process and predicting the storage stability.