Background: Pseudomonas aeruginosa (PsA) is an opportunistic bacterium associated with chronic respiratory infections in people with cystic fibrosis (pwCF). PsA clonal lineages persist in pwCF despite the clinical benefits of CFTR modulators, warranting the identification of novel anti-PsA agents. Peptidyl-arginine deiminases (PADs) enzymes catalyse the post-translational modification of peptidyl-arginine to peptidyl-citrulline. Neutrophils express PAD2 and PAD4, but their known activity is limited to histone citrullination. This study aimed to explore the anti-PsA properties of neutrophil-derived and recombinant PAD enzymes. Methods: Bactericidal and biofilm inhibition properties of recombinant PADs were assessed against PsA PAO1 and CF multidrug resistant isolate PGO2330. Results: PAD2 and PAD4 were localised to neutrophil phagocytic vacuoles following degranulation from primary granules. In phagosomal killing assays, inclusion of the PAD inhibitors, AFM-30a (p=0.05) or GSK484 (p=0.0079) increased PAO1 survival. In vitro, PAD2 and PAD4 bound to the outer membrane of PAO1 and within 30 min, reduced survival to 50.3±6.7% (p=0.0006) and 43.4±2.0% (p=0.0005), respectively. Additionally, PAD2 and PAD4 exhibited biofilm inhibition IC₅₀ values of 6.6±4.9 and 16.6±11.0 nM, and 13.0±11.1 and 14.9±6.3 nM, against PAO1 and PGO2330, respectively. PAD2 and PAD4 reduced PAO1 las, rhl and pqs quorum sensing systems (p<0.0021). Conclusion: PADs are involved in neutrophil phagosomal killing of PsA, and recombinant PADs possess anti-microbial and anti-biofilm properties against PsA PAO1 and MDR CF isolate PGO2330, supporting research into their clinical applications. Funding: IRC (GOIPG/2023/3100).