The McMurdo Dry valleys (DV) in Antarctica are one of the harshest environments on Earth, with high levels of UV radiation, freeze-thaw cycles, low moisture, and nutrient content. Bacteria in this extreme environment have adapted to slow growth and low-nutrient conditions. It is proposed that bacteria from Antarctic DV systems may possess unique DNA repair enzymes and pathways that enable their survival in such extreme conditions. Exploring these DNA repair systems may offer valuable insights into microbial survival in extreme environments or even extra-terrestrial settings. To investigate this, DNA repair enzymes were identified from Antarctic DV microbial metagenomes using in silico analysis and in vivo characterisation of recombinantly produced proteins. Four candidate enzymes were chosen for structural and biological characterisation. Three of these enzymes are characterised as LigB type ATP-dependent DNA ligases, with the typical arrangement of a DNA binding domain, adenylation domain and an OB-fold domain. These ligases can utilise both ATP and ADP nucleotide cofactors for the ligation of nick and mismatch DNA substrates, requiring the addition of magnesium or manganese metal ions. One LigB ligase stands out due to its interesting fusion with an N-terminally located nuclease domain, which resembles an extensively studied MBL-β-CASP domain found in all domains of life. The recombinantly expressed nuclease domain can coordinate several types of metal ions and shows nuclease activity against a diverse range of DNA substrates. Nuclease activity favoured single-stranded DNA substrates, in a 5’ to 3’ direction, and was particularly active on substrates with abasic sites or 5’ flaps. The fourth protein was initially annotated as a hypothetical protein, however in silico analysis revealed that it exhibited homology to NucS nucleases identified in archaea and bacteria. This NucS homolog is a monomeric enzyme, made up of four domains, which is different from other proteins in this family. This enzyme shows a preference for single-stranded DNA and has a broad range of nuclease activity on damaged and mismatched DNA substrates. It can utilise both magnesium and manganese metal ions for activity on DNA substrates. Overall, these findings suggest that the described enzymes will play a role in DNA repair pathways, potentially in high-damage environments like the DVs of Antarctica.