The availability of water, which can be expressed in terms of water activity (aw), is one of the most important determinants for microbial homeostasis and growth on surface to air interfaces. Here we show, using an environmental control chamber containing a precisely controlled temperature/aw gradient in combination with a mathematical approach, that the environmental aw growth limit of a microorganism can be lower than its intracellular aw limit. This internal limit represents the point at which microbial cells cannot lower their internal aw any further in response to low external aw values without interfering with essential intracellular processes. To grow at external aw values below their internal limit, microbes need to generate more water metabolically than they lose to their environment. This internal aw limit can be calculated by measuring the aw growth limit of an organism at different water vapour diffusivities using barometric pressure as a variable. Fascinating morphological changes, such as rope-like superstructures formed by B. subtilis, are furthermore observed in response to low external aw values in particular around the calculated intracellular aw limit. The intracellular aw limit of an organism is a decisive parameter for water limitation-induced adaptations in cellular hydrophilicity and morphogenesis.