Bacterial riboswitches are structured RNAs that bind small metabolites to control downstream gene expression. Two riboswitch classes have been reported to sense nicotinamide adenine dinucleotide (NAD+), which plays a key redox role in cellular metabolism. The NAD+-I (class I) riboswitch stands out because it comprises two homologous, tandemly arranged domains. However, previous studies examined the isolated domains rather than the full-length riboswitch. Crystallography and ligand binding analyses led to the hypothesis that each domain senses NAD+ but with disparate equilibrium binding constants (KD) of 127 μM (domain I) and 3.4 mM (domain II). Here, we analyzed individual domains and the full-length riboswitch by isothermal titration calorimetry to quantify the cofactor affinity and specificity. Domain I senses NAD+ with a KD of 24.6 ± 8.4 μM but with a reduced ligand-to-receptor stoichiometry, consistent with nonproductive domain self-association observed by gel-filtration chromatography; domain II revealed no detectable binding. By contrast, the full-length riboswitch binds a single NAD+ with a KD of 31.5 ± 1.5 μM; dinucleotides NADH and AP2-ribavirin also bind with one-to-one stoichiometry. Unexpectedly, the full-length riboswitch also binds a single ATP equivalent (KD = 11.0 ± 3.5 μM). The affinity trend of the full-length riboswitch is ADP = ATP > NAD+ = AP2-ribavirin > NADH. Although our results support riboswitch sensing of a single NAD+ at concentrations significantly below the intracellular levels of this cofactor, our findings do not support the level of specificity expected for a riboswitch that exclusively senses NAD+. Gene regulatory implications and future challenges are discussed.