Mycoplasma hyopneumoniae is the cause of porcine Enzootic Pneumonia, a chronic respiratory disease of significant worldwide importance in pig production. Mycoplasma spp have evolved with a reduction in gene content and their tiny genomes lack many biosynthetic pathways, such as those for de novo nucleotide synthesis. Therefore they are reliant on the host for nucleotides, and expression of membrane-associated nucleases may be an important means of scavenging them from their environment. M. hyopneumoniae 232 (Mh232) has at least two nucleases: membrane nuclease mnuA (mhp597) and mhp379. The latter has been well-characterised and is part of an operon involved in the acquisition of nucleotides. We sought to isolate transposon insertion mutants of both nucleases using a “haystack mutagenesis” approach, and to derive targeted gene disruptions by homologous recombination. It was not possible to isolate mhp379 mutants, suggesting that either it is essential for in vitro growth, or that disruption has polar effects on other genes in the operon. Targeted disruptions and transposon mutants were isolated in mnuA and showed no change in in vitro growth characteristics. Using lambda phage DNA and plasmid DNA as substrates, mnuA mutants displayed a remarkable reduction in nuclease activity compared to wild-type Mh232. This reduction in nuclease activity was also shown using a plate-based DNA agar developed for Mh232. It appears that cell surface expressed nuclease activity of Mh232 comes predominantly from mnuA rather than mhp379, but that mnuA is not essential for optimal in vitro culture. It is possible that mnuA expression provides a growth advantage in vivo in a more hostile environment. Alternatively, mnuA may be associated with evasion of host immune responses in allowing the degradation of neutrophil extracellular traps and avoidance of phagocytosis. Such mechanisms have been proposed for other bacteria such as Staphylococcus aureus that also express potent cell-surface nuclease activity.