Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus are acidophilic Gram-negative -proteobacteria involved in the bioleaching of metal sulfides. Bacterial attachment to mineral surface and biofilm development play a pivotal role in this process. Therefore, the understanding of biofilm formation has relevance to the design of biological strategies to improve the efficiency of bioleaching processes. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in biomining bacteria. In many bacteria, the intracellular level of c-di-GMP molecules regulates the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development. Thus, we recently started the study of c-di-GMP pathway in biomining bacteria. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). So far, two kinds of effectors have been identified, including three protein families (pilZ, PleD and FleQ) and a conserved RNA domain (GEMM) which acts as a riboswitch. We previously reported the existence of different molecular players involved in c-di-GMP pathway in A. ferrooxidans ATCC 23270. Here, we expanded our work to other Acidithioibacillus species: A. thiooxidans ATCC 19377 and A. caldus ATCC 51756. In both, we identified several putative-ORFs encoding DGC, PDE and effector proteins. By using total RNA extracted from A. ferrooxidans and A. caldus cells in RT-PCR and qPCR experiments, we demonstrated that these genes are expressed. In addition, we characterized the presence of c-di-GMP in A. ferrooxidans ATCC 23270 and A. caldus ATCC 51756 cell extracts. Taken together, these results strongly suggest that A. ferrooxidans, A. caldus and A. thiooxidans possess functional c-di-GMP pathways. As it occurs in other Gram-negative bacteria, this pathway should be involved in the regulation of the planktonic/biofilm switch. In the future, we have to integrate this new biological dimension to improve the biological understanding of bioleaching.