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 local fatty acid sensing regulators. To understand the relative contribution of the desaturases in defining the A. baumannii lipidome, individual deletion derivatives were examined for their ability to persist across a range of physiologically relevant growth conditions, including osmotic pressure and nutrient depletion. The fatty acid composition and the subsequent biophysical properties of the membrane were also determined. 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.