Microbial activity has been documented in various lacustrine environments, suggesting its fundamental role in mineral precipitation and, therefore, in the formation of organo-deposits such as microbialites. Many studies are currently focused on documenting how the association of microbes and extracellular polymeric substances (EPSs) may influence the authigenesis of Mg-rich clay minerals and the subsequent carbonate precipitation in growing microbialites in lacustrine environments. In this study, we investigate the present-day microbialites of the alkaline Bagno dell’Acqua lake (Pantelleria Island, Italy) using X-ray diffraction (XRD) scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Our results reveal the intimate association of Mg-smectite/carbonate minerals with the EPS and microbes, and, for the first time, we selected microbes belonging to phylum Firmicutes (Bacillus sp.), from natural microbialites, to carry out laboratory experiments that testify their direct role in the precipitation of clay and carbonate minerals.