Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals, including those with cystic fibrosis. P. aeruginosa extensively forms biofilms which significantly contribute to its growth and persistence in a wide range of environments. In addition, its success can be attributed to several secreted proteases. Here, we investigated the aminopeptidase PaAP from P. aeruginosa, which is one of the most abundant extracellular proteins in the biofilm matrix. Increasing evidence suggests this quorum-sensing-regulated peptidase is associated with biofilm development and contributes to nutrient recycling by complementing the activity of other secreted proteases. We confirmed that post translational processing was required for activation, as a C-terminal truncation mutant possessed ~100-fold higher activity than the full-length enzyme. We determined that PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. High-resolution crystal structures of wild type enzyme and mutants revealed the mechanism of autoinhibition, whereby the Cterminal pro-peptide locks the protease-associated (PA) domain and the catalytic peptidase domain, into a closed, inhibited conformation. Inspired by this selfregulatory mechanism and guided by structural data, we designed a highly potent small cyclic-peptide inhibitor (KI in the nM range). This inhibitor recapitulates the deleterious phenotype observed with a PaAP deletion mutant in liquid cultures and biofilm assays and presents a path towards targeting secreted proteins in a biofilm context.