AbstractSmall non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Despite the identification of hundreds of bacterial sRNAs, their roles on bacterial physiology and virulence remain largely unknown, as is the case of bacteria of theBurkholderia cepaciacomplex (Bcc). Bcc is a group of opportunistic pathogens with relatively large genomes that can cause lethal lung infections amongst cystic fibrosis (CF) patients. To characterise sRNAs expressed by Bcc bacteria when infecting a host, the nematodeCaenorhabditis eleganswas used as an infection model by the epidemic CF strainB. cenocepaciaJ2315. A total of 108 new and 31 previously described sRNAs with a predicted Rho independent terminator were identified, most of them located on chromosome 1. RIT11b, a sRNA downregulated underC. elegansinfection conditions, was shown to directly affectB. cenocepaciavirulence, biofilm formation, and swimming motility. RIT11b overexpression reduced the expression of the direct targetsdusAandpyrC, involved in biofilm formation, epithelial cell adherence, and chronic infections in other organisms. The in vitro direct interaction of RIT11b with thedusAandpyrCmessengers was demonstrated by electrophoretic mobility shift assays. To the best of our knowledge this is the first report on the functional characterization of a sRNA directly involved inB. cenocepaciavirulence.Key points• 139 sRNAs expressed by B. cenocepacia during C. elegans infection were identified• The sRNA RIT11b affects B. cenocepacia virulence, biofilm formation, and motility• RIT11b directly binds to and regulates dusA and pyrC mRNAsSupplementary InformationThe online version contains supplementary material available at 10.1007/s00253-023-12530-3.