Host-microbe communication via small molecule signals is often poorly understood at the molecular level. Under conditions of host stress, levels of the human opioid peptide dynorphin are elevated, triggering virulence in the opportunistic pathogenic bacterium Pseudomonas aeruginosa (PAO1) via an unknown pathway. Here we apply multiple chemical biology strategies to unravel the mode of action of this putative interkingdom signal. We designed and applied dynorphin-inspired photoaffinity probes (DYN4 and DYN5) to reveal the protein targets of the peptide in live bacteria via chemical proteomics. Sensor kinase ParS was identified as a potential hit. Subsequent full proteome studies revealed that dynorphin(1-13) (DYN) induces an antimicrobial peptide-like response in Pseudomonas, with specific upregulation of membrane defence mechanisms (datasets Stat.10 and Exp.5). No such response was observed in a parS mutant (Stat.1), functionally linking DYN engagement with ParS to this phenotype.