The Ribonuclease III (RNase III) enzymatic family is implied in many important biological processes from the bacteria until humans. In this sense, they have been selected as drug-target candidates for drug development. Although RNase III members show a high degree of sequence diversity, they generally share common structural and some degree of enzymatic activity. The accessory domains are key determinants for both the substrate selectivity and the biological function of each RNase III type, however, their role is still under study. The in vitro enzymatic activity of three RNase III members from class I (E. coli RNase III, Schizosaccharomyces pombe Pac1 and Saccharomyces cerevisiae Rntp1) and the human Drosha placed in class II was tested against four different substrates. These two RNase III classes encompass members having different domain organization. Enzymatic activity differences were found among members of the class I, which get increased when the human Drosha (class II) was included in the test. The substrate promiscuity of the E. coli RNase III was corroborated in vivo through its expression in S. cerevisiae, as reported previously, but was extended here to Pichia pastoris. The new biochemical data integrated with previous studies confirmed that RNases III substrate specificity is highly influenced by its protein structure architecture. This fact also allowed drawing evolutionary links between RNase III members from their structural and substrate specificity differences. More details about the results can be consulted at DOI: 10.2174/1381612822666160511150416.