Acinetobacter baumannii is a Gram-negative nosocomial pathogen associated with significant morbidity and mortality in susceptible individuals. A number of persistence and resistance strategies contribute to the success of this organism, including an ability to alter the biophysical properties of the membrane in response to changing environmental conditions. This process is achieved, in part, by the fatty acid and phospholipid biosynthetic pathways. However, the molecular basis and the interplay of these lipid homeostasis mechanisms in A. baumannii is largely ill-defined. We have identified critical roles of two related, but functionally distinct desaturases in unsaturated fatty acid production and defined that these are co-ordinately regulated by fatty acid sensing regulators. The two major negatively-charged phospholipid species present in the A. baumannii membrane are phosphatidylglycerol and cardiolipin, with their biosynthesis relying upon the phosphatidylglycerolphosphatases PgpA and PgpB. We found that pgpB is co-transcribed with adeIJK, a tripartite efflux pump which has recently identified to be involved in lipid extrusion, inferring a further association with lipid homeostasis. To understand the relative contribution of the desaturases, PgpA/B and the AdeIJK efflux system in defining the A. baumannii lipidome, individual deletion derivatives were examined for their fatty acid and phospholipid composition and the subsequent biophysical properties of the membrane. Given the essentiality of fatty acid and phospholipid biosynthesis in bacterial viability, and their critical role in stress adaptation, investigations into the mechanisms of lipid homeostasis are crucial for understanding how this pathogen of global significance adapts to host-induced and environmental stress.