Bioinformatic approaches are described for the discovery of small regulatory RNAs (srRNAs) in the biomining microorganism Acidithiobacillus ferrooxidans. Intergenic regions of the annotated genome were extracted and computationally searched for srRNAs. Candidate srRNAs that were associated with predicted sigma 70 promoters and/or rho-independent terminators were chosen for further study. Experimental validation is presented for 6S srRNA and frr. srRNAs are known to control gene expression in a wide variety of microorganisms, usually at the post-transcriptional level, by acting as antisense RNAs that bind targeted mRNAs or by interacting with regulatory proteins. srRNAs are involved in the regulation of a large variety of processes. Frr is an RNA antisense to fur; the latter encodes a global regulator involved the control of a large number of genes involved in iron uptake and homeostasis. Because of the widespread occurrence and extensive repertoire of regulatory functions afforded by srRNAs, it is expected that their discovery functional analysis in biomining microorganisms will contribute to improving our understanding of the microbiology of bioleaching processes.