Microbialites are organo‐sedimentary structures formed throughout most of the Earth history, over a wide range of geological contexts, and under a multitude of environmental conditions affecting their composition. The carbon and oxygen isotope records of carbonates, which are most often their main constituents, have been used as a widespread tool for paleoenvironmental reconstructions. However, the multiplicity of factors that influence microbialites formation is not always properly distinguished in their isotopic record, in both ancient and modern settings. It is therefore crucial to refine our understanding of the processes controlling microbialites isotopic signal. Here, we analyzed the carbon and oxygen isotope compositions from bulk and micro‐drilled carbonates as well as bulk organic carbon isotope compositions in microbialites from four Mexican volcanic crater lakes of increasing alkalinity. The survey of four lakes allows comparing microbialite formation processes and their geochemical record within distinct physico‐chemical contexts. The geochemical analyses were performed in parallel to petrographic and mineralogical characterization and interpreted in light of the known microbial community composition for microbialites of the same lakes. Combining these data, we show that the potential for isotopic biosignature preservation primarily depends on physico‐chemical conditions. Carbon isotope biosignatures pointing out to an autotrophic influence on carbonate precipitation are preserved in the lowest alkalinity lakes. By contrast, higher alkalinity lakes, where microbialites are more massive, favor carbonate precipitation in isotopic equilibrium with the lake water, with secondary influence of heterotrophic organic carbon degradation. From these results, we suggest that microbialite carbonate C isotope records can be interpreted as the balance between the microbialite net primary productivity and the amount of precipitation that relates to physico‐chemical forcing. The signals of microbialite oxygen isotope compositions highlight a lack of understanding in the oxygen isotope records of relatively rare carbonate phases such as hydromagnesite. Nonetheless, we show that these signals are primarily influenced by the basins' hydrology, though biological effects may also play a (minor) role. Overall, both carbon and oxygen isotopic signals may record a mixture of different local/global and biotic/abiotic phenomena, making microbialites intricate archives of their growth environment, which should thus be interpreted with cautions and in the light of their surrounding sediments.