Streptococcus pyogenes nuclease A (SpnA) and streptococcal 5' nucleosidase A (S5nA) are two recently described virulence factors from the human pathogen S. pyogenes. In vitro studies have shown that SpnA is a nuclease that cleaves ssDNA and dsDNA, including the DNA backbone of neutrophil extracellular traps. S5nA was shown to hydrolyse AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. S5nA also generates the macrophage-toxic deoxyadenosine from dAMP. However, detailed in vivo studies of the two enzymes have been hampered by difficulties with using current animal models for this exclusive human pathogen. Here we report the identification of two novel enzymes from the fish pathogen Streptococcus iniae that show similarities to SpnA and S5nA in amino acid sequence, protein domain structure and biochemical properties. We propose that SpnAi and S5nAi are orthologues of the S. pyogenes enzymes, providing a rationale to analyse the in vivo function of the two enzymes using a S. iniae-zebrafish infection model.