The opportunistic pathogen Acinetobacter baumannii is commonly associated with hospital-acquired bacteremia and respiratory infections. Rapidly emerging antibiotic resistance in A. baumannii poses such a risk that the World Health Organisation has listed it as a priority one critical organism where new antibiotics are urgently required. Many A. baumannii strains express a type VI secretion system (T6SS). This molecular machine delivers antibacterial toxins directly into adjacent bacteria, which provides a competitive advantage in certain niches. T6SS positive cells also express cognate immunity proteins that bind and neutralise effectors, protecting self and sibling cells. One such effector-immunity pair from a clinical A. baumannii strain (AB307-0294) is the type VI DNase effector 16 (Tde16) and its cognate DNase immunity protein (Tdi16). To confirm activity of Tde16, attempts were made to express and purify the DNase domain in E. coli; however, cells expressing the domain were not viable. Therefore, guided by homology, a double alanine mutant at the predicted active site was constructed (AHH to AAA) to produce Tde16AAA. Tde16AAA was non-toxic when expressed in E. coli and subsequent purification of Tde16AAA revealed it was monomeric and bound DNA. AlphaFold modelling of Tde16AAA revealed a positively charged surface, suggesting that DNA binding occurs using electrostatic interactions. Mutation of the predicted DNA binding site reduced the binding affinity of Tde16AAA to DNA, confirming that binding is mediated through electrostatic interactions. Purification of the cognate immunity protein, Tdi16, revealed the two proteins directly interact with a binding stoichiometry of 1:1 and that presence of Tdi16 disrupts Tde16AAA DNA binding. Further characterisation of these effector/immunity interactions could allow for manipulation and treatment of A. baumannii infections in the future. Exploration of novel effectors alone may also elucidate antimicrobial targets to aid in combatting resistance.