Biocrusts are a worldwide phenomenon, contributing substantially to ecosystem functioning. Their growth and survival depend on multiple environmental factors, including climatic ones, and the relations of these factors to physiological processes. Responses of biocrusts to individual environmental factors have been examined in a large number of field and laboratory experiments. These observational data, however, have rarely been assembled into a comprehensive, consistent framework that allows quantitative exploration of the roles of multiple environmental factors and physiological properties for the performance of biocrusts, in particular across climatic regions. Here we used a data-driven mechanistic modelling framework to simulate the carbon balance of biocrusts, a key measure of their growth and survival. We thereby assessed the relative importance of physiological and environmental factors for the carbon balance at six study sites that differ in climatic conditions. Moreover, we examined the role of seasonal acclimation of physiological properties using our framework, since the effects of this process on the carbon balance of biocrusts are poorly constrained so far. We found substantial effects of air temperature, CO2 concentration, and physiological parameters that are related to respiration on biocrust carbon balance, which differ, however, in their patterns across regions. The ambient CO2 concentration is the most important factor for biocrusts from drylands, while air temperature has the strongest impact at alpine and temperate sites. Metabolic respiration cost plays a more important role than optimum temperature for gross photosynthesis at the alpine site; this is not the case, however, in drylands and temperate regions. Moreover, we estimated a small annual carbon gain of 1.5 g m−2 yr−1 by lichen-dominated biocrust and 1.9 g m−2 yr−1 by moss-dominated biocrust at a dryland site, while the biocrusts lost a large amount of carbon at some of the temperate sites (e.g. −92.1 for lichen-dominated and −74.7 g m−2 yr−1 for moss-dominated biocrust). These strongly negative values contradict the observed survival of the organisms at the sites and may be caused by the uncertainty in environmental conditions and physiological parameters, which we assessed in a sensitivity analysis. Another potential explanation for this result may be the lack of acclimation in the modelling approach, since the carbon balance can increase substantially when testing for seasonally varying parameters in the sensitivity analysis. We conclude that the uncertainties in air temperature, CO2 concentration, respiration-related physiological parameters, and the absence of seasonal acclimation in the model for humid temperate and alpine regions may be a relevant source of error and should be taken into account in future approaches that aim at estimating the long-term biocrust carbon balance based on ecophysiological data.