Acinetobacter baumannii causes severe disease in immunocompromised people including respiratory and urinary tract infections. Emergence of antibiotic-resistant strains has severely reduced therapeutic options and new control strategies are urgently needed. A. baumannii possess a type VI secretion system (T6SS) that is involved in interbacterial competition and can give the bacteria a competitive advantage in multispecies environments. The regulation mechanism of the A. baumannii T6SS system is unknown but T6SS regulatory pathways have been identified in other species, including Pseudomonas aeruginosa and Agrobacterium tumefaciens. To characterise T6SS regulatory pathways in A. baumannii, we used bioinformatics to identify putative T6SS regulators based on homology with known/predicted T6SS regulators from other species. We then made deletion mutants and overexpression strains representing each gene. T6SS activity of the recombinant strains was assessed by measuring the secretion levels of the T6SS needle protein Hcp using western blot, and by observing T6SS-mediated killing of E. coli by co-culturing recombinant A. baumannii predator strains with E. coli prey. Two A. baumannii proteins, FhaA and Pkn5 were predicted to be homologues of P. aeruginosa proteins involved in a threonine phosphorylation (TPP) -dependent T6SS regulatory pathway but deletion nor overexpression of these genes/proteins in A. baumannii altered T6SS activity, suggesting that the T6SS is not regulated in a TPP-dependent manner. However, overexpression of the A. baumannii proteins TagF and ABBFA_02194, homologues of predicted T6SS regulatory proteins produced by Acinetobacter baylyi, caused complete loss or suppression of T6SS activity, respectively. Transcriptomic analysis indicated this was not via a transcriptional mechanism. Finally, we showed that TagF and ABBFA_02194 are both required to fully repress T6SS in A. baumannii. These results suggest the regulation of the T6SS in Acinetobacter involves a novel pathway and may indicate new therapeutic targets for reducing survival of A. baumannii in multispecies environments.