Burkholderia cenocepacia, is a Gram-negative bacterial species belonging to the Burkholderia cepacia complex (Bcc), a group of closely related species found ubiquitously in the environment that are also opportunistic human pathogens. B. cenocepacia is predicted to be able to secrete proteins through the type six secretion system (T6SS). Characterised in other bacteria, this secretory-apparatus can perforate the cellular membrane in pro- and/or eukaryotic targets, to deliver an arsenal of effector proteins. A cognate immunity protein is required to provide protection against these effectors. Identifying and characterising the T6SS-secreted effectors, their cognate immunity proteins and their molecular targets is key to understanding the role the T6SS plays in the interactions between B. cenocepacia and its target organism(s). Here, this study defines a functional role for the B. cenocepacia T6SS in bacterial competition. Moreover, this is partly dependent on the phospholipase A1 activity of a novel evolved TssI, TssI0667. The involvement of the B. cenocepacia T6SS in pathogenicity in two models of innate immune infection were assessed, but these studies did not demonstrate a contribution from this system to overall virulence. A further putative T6SS-dependent effector, TanBH , was characterised as having nuclease activity but the role of this candidate effector in vivo remains unclear. The cognate immunity protein for each effector was identified as Tli6 and TaaB1H , respectively, and demonstrated to form a direct complex with its effector that inhibited the toxic activity of the latter. Therefore, this work has begun to uncover mechanisms used by Bcc bacteria, such as B. cenocepacia, that may contribute towards their survival in their natural environment but has not shed any further light on the role of the T6SS in the pathogenic strategies of these bacteria.