ABSTRACT Gene expression profiles provide insight into how microorganisms respond to changing environmental conditions. However, few studies have integrated expression profile analyses of both coding genes and non-coding RNAs (ncRNAs) to characterize the functional activity of microbial community members. Here, we defined gene expression profiles from environmental and laboratory-grown acidophilic biofilms using RNASeq. In total, 15.8 million Illumina reads were mapped to the genomes of 26 acidophilic microorganisms and nine viruses reconstructed from the Richmond Mine at Iron Mountain, California. More than 99% of the genome was transcribed in three Leptospirillum species, and > 80% in the archaea G-plasma and Ferroplasma Type II. High gene expression by G-plasma and the Leptospirillum Group II UBA strain correlated with extremely acidic conditions, whereas high transcriptional expression of Leptospirillum Group III and Leptospirillum Group II 5way-CG strain occurred under higher pH and lower temperature. While expression of CRISPR Cas genes occurs on the sense strand, expression of the CRISPR loci occurs on the antisense strand in the Leptospirilli . A novel riboswitch associated with the biosynthetic pathway for the osmolyte ectoine was upregulated when each specific Leptospirillum Group II strain was growing under the conditions most favorable for it. Newly described ncRNAs associated with CO dehydrogenase (CODH) suggest regulation of expression of CODH as a CO sensor in mature biofilms in the Leptospirilli . Results reveal the ways in which environmental conditions shape transcriptional profiles of organisms growing in acidophilic microbial communities and highlight the significance of ncRNAs in regulating gene expression. IMPORTANCE Microorganisms play important roles in environmental acidification and in metal-recovery based bioleaching processes. Therefore, characterizing how actively growing microbial communities respond to different environments is key to understanding their role in those processes. Microorganisms express their genes, both coding and non-coding, differently depending on environmental factors, thus evaluating community expression profiles inform about the ecology of actively growing microorganisms. Here we used community transcriptomic analyses to characterize gene expression profiles from biofilm communities growing under extremely acidic conditions. Results expand our knowledge of how acidophilic microorganisms respond to changes in their environment and provide insight into possible gene regulation mechanisms.