Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as nucleotide second messengers. Recent studies have identified functional roles for the non-canonical 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) in both eukaryotes and prokaryotes. In , 2′,3′-cNMPs are produced by RNase I-catalyzed RNA degradation, and these cyclic nucleotides modulate biofilm formation through unknown mechanisms. The present work dissects cellular processes in and Typhimurium that are modulated by 2′,3′-cNMPs through the development of cell-permeable 2′,3′-cNMP analogs and a 2′,3′-cyclic nucleotide phosphodiesterase. Utilization of these chemical and enzymatic tools, in conjunction with phenotypic and transcriptomic investigations, identified pathways regulated by 2′,3′-cNMPs, including flagellar motility and biofilm formation, and by oligoribonucleotides with 3'-terminal 2′,3′-cyclic phosphates, including responses to cellular stress. Furthermore, interrogation of metabolomic and organismal databases has identified 2′,3′-cNMPs in numerous organisms and homologs of the metabolic proteins that are involved in key eukaryotic pathways. Thus, the present work provides key insights into the roles of these understudied facets of nucleotide metabolism and signaling in prokaryotic physiology and suggest broad roles for 2′,3′-cNMPs among bacteria and eukaryotes. Bacteria adapt to environmental challenges by producing intracellular signaling molecules which control downstream pathways and alter cellular processes for survival. Nucleotide second messengers serve to transduce extracellular signals and regulate a wide array of intracellular pathways. Recently, 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) were identified for contributing to the regulation of cellular pathways in eukaryotes and prokaryotes. In this study we define previously unknown cell processes that are affected by fluctuating 2′,3′-cNMP levels or RNA oligomers with 2′,3′-cyclic phosphate termini in and Typhimurium, providing a framework for studying novel signaling networks in prokaryotes. Furthermore, we utilize metabolomics databases to identify additional prokaryotic and eukaryotic species that generate 2′,3′-cNMPs as a resource for future studies.