Natural or industrial pollution of source water by arsenic poses a serious threat to the health of tens of millions of people worldwide, while limited data have addressed its influence on the waterborne pathogens. This study examines the effects of arsenic exposure on Pseudomonas aeruginosa, an opportunistic pathogen that poses a health hazard in public water supplies. Our findings reveal that arsenic exposure at concentrations of 0.12–20 mg/L As(III) causes rapid growth arrest in P. aeruginosa. This exposure also leads to a marked decrease in elastase, pyocyanin, and flagellar production, while significantly increasing siderophore and extracellular polysaccharide production. Proteomic analysis indicates that both low (0.12 mg/L) and high (1.2 mg/L) arsenic levels trigger an antioxidant defense response, evidenced by the upregulation of Fnr-2, TrxB2, Ohr, AlgZ, and PslG. Concurrently, proteins related to basal metabolism, quorum sensing, T3SS, pyocyanin biosynthesis, and flagellar assembly are downregulated. Moreover, arsenic exposure enhances the expression of proteins involved in siderophore biosynthesis and transporters associated with iron uptake and multidrug resistance. In vitro assays further demonstrate that arsenic exposure reduces bacterial cytotoxicity while promoting survival and proliferation under antibiotic treatment. In summary, this study provides comprehensive insights into how arsenic affects bacterial virulence and antibiotic tolerance in P. aeruginosa, highlighting the potential public health risks posed by the frequent co-occurrence of arsenic-contaminated water sources and multidrug-resistant bacteria.