Here, we have investigated, for the first time, the serine, threonine and tyrosine phosphoproteome in the extracellular medium of A. baumannii during biofilm mode of growth for two strains: the reference strain ATCC 17978 and the virulent multi-drug resistant strain AB0057, a clinical isolate from a bloodstream infection. Overall, a total of 109 and 146 phosphorylated proteins were identified for ATCC 17978 and AB0057, respectively. We also showed that one specific phosphorylation site was essential for the activity of the A. baumannii type VI secretion system. This provides the first global phosphosecretome profiling of A. baumannii. This analysis represents a promising starting point for further investigations on the biological role of phosphorylation in A. baumannii.