Acinetobacter baumannii is a Gram-negative opportunistic pathogen responsible for a range of nosocomial and community-acquired infections. The rapidly increasing prevalence of multi-drug resistant (MDR) A. baumannii strains, and other MDR bacterial pathogens, raises serious concerns about the availability of antimicrobial treatments for these organisms. Notably, many Gram-negative bacteria utilise a type VI secretion system (T6SS), which structurally resembles an inverted bacteriophage tail, to deliver toxic effector proteins directly into target cells. A. baumannii uses the T6SS to attack competing bacteria by delivering a diverse array of antibacterial effector proteins into nearby target cells. Typically, T6SS-producing species are protected from sibling attack via the presence of specific immunity proteins. However, preliminary data suggested that the clinical isolate A. baumannii AB307-0294 may be immune to sibling-mediated T6SS killing in the absence of these specific immunity proteins. Thus, we hypothesised that A. baumannii AB307-0294 uses a self-recognition mechanism to prevent T6SS targeting of sibling cells. We used directed mutagenesis to delete all three T6SS effector/immunity gene pairs from A. baumannii AB307-0294. This effector/immunity mutant demonstrated significantly reduced survival when co-cultured with wild-type A. baumannii AB307-0294, compared to co-culture with a T6SS-inactive A. baumannii AB307-0294 strain (p=0.0286, Mann-Whitney), indicating that A. baumannii AB307-0294 attacks sibling cells via the T6SS. Complementation of the effector/immunity mutant with the three immunity genes prevented this T6SS-mediated killing (p=0.3429, Mann-Whitney). Interestingly, we did not observe complete killing of the effector/immunity gene mutant, as would be expected in the absence of the required T6SS immunity proteins, suggesting that additional defence mechanisms may be involved in protection against the T6SS. As such, we are employing Transposon Directed Insertion Sequencing (TraDIS) to screen for defence mechanisms that may protect A. baumannii AB307-0294 from sibling T6SS-mediated killing in the absence of immunity proteins. Characterisation of T6SS defence mechanisms provides insight into the dynamics of T6SS killing and may facilitate the use of this system as a novel antimicrobial delivery method in future.