The synthesis and degradation of RNA is a fundamental and essential process for all life forms. It is imperative that cells utilize multiple mechanisms to modulate the lifetimes of RNA molecules to ultimately control protein synthesis, to maintain homeostasis or adapt to environmental challenges. One mechanism to directly alter the stability or half-life of an RNA is through the direct enzymatic activity of ribonucleases (RNases). Bacterial organisms encode for many different RNases that possess distinct functions in RNA metabolism. It is through the actions of multiple cellular RNases that a long RNA polymer corresponding to an mRNA, rRNA, tRNA, or sRNA can be fully degraded back into nucleotide monophosphate precursors. The nucleoside monophosphate precursors are recognized by kinases that ultimately recycle these molecules for use in the synthesis of other long RNA polymers. The processing of RNA polymers by endo- and exoribonucleases generates nucleoside monophosphates as well as short RNA oligonucleotides ranging from 2-6 nucleotides in length. Previously, a subset of enzymes broadly referred to as “nanoRNases” were found to process these short RNA fragments. Oligoribonuclease (Orn), NanoRNase A (NrnA), NanoRNase B (NrnB), and NanoRNase C (NrnC) were previously ascribed the function of indiscriminately processing nanoRNA substrates. However, recent analyses of the evolutionarily related DnaQ-fold containing proteins Orn and NrnC have provided compelling evidence that some nanoRNase protein families possess distinct dinucleotide substrate length preferences (Kim et al., 2019; Lormand et al., 2021). To determine whether all nanoRNases are diribonucleotide-specific enzymes, we utilized a combination of in vitro and in vivo assays to conclusively elucidate the substrate specificity and intracellular roles of the DHH-DHHA1 family proteins NrnA and NrnB. Through an in vitro biochemical survey of many NrnA and NrnB protein homologs, including from organisms of varying degrees of relatedness, we have determined that there are many functional dissimilarities contained within the DHH-DHHA1 protein family. Furthermore, we have conducted a rigorous investigation into the biological and biochemical functions of NrnA and NrnB in the Gram-positive model organism Bacillus subtilis. These analyses have shown that B. subtilis NrnA and NrnB are not redundant in biochemical activities or intracellular functions, as previously believed. In fact, we have found that B. subtilis NrnA is a 5’-3’ exoribonuclease that degrades short RNAs 2-4 nucleotides in length during vegetative growth, while B. subtilis NrnB is specifically expressed within the developing forespore and functions as a 3’-5’ exoribonuclease that processes short RNA in addition to longer RNA substrates (>40-mers). Our collective data provide a strong basis for the subdivision of the DHH-DHHA1 protein family on the basis on their diverse substrate preferences and intracellular functions.