Arsenic contamination of water sources, whether from natural or industrial origins, represents a significant risk to human health. However, its impact on waterborne pathogens remains understudied. This research explores the effects of arsenic exposure on the opportunistic pathogen Pseudomonas aeruginosa, a bacterium found in diverse environments. The arsenic exposure at concentrations of 0.12-20 mg/L As(III) resulted in rapid growth arrest of P. aeruginosa. Moreover, arsenic exposure significantly reduced the production of key virulence factors such as elastase (by 1.48- to 9.24-fold), pyocyanin, and flagella while increasing siderophore and extracellular polysaccharide production (by 1.44-1.75 and 1.36-2.59 times, respectively). Proteomic analysis revealed that both low (0.12 mg/L) and high (1.2 mg/L) As(III) levels activated an antioxidant defense response, with upregulation of Fnr-2, TrxB2, and Ohr. Furthermore, arsenic-induced the overexpression of multidrug resistance efflux proteins MexAB-OprM, MexCD-OprJ, and MexEF-OprN. At the same time, proteins associated with quorum sensing (QS), type III secretion system (T3SS), pyocyanin biosynthesis, and flagellar assembly were downregulated. In vitro assays confirmed that arsenic reduced bacterial virulence and significantly enhanced survival and proliferation under antibiotic treatment. These results indicate that arsenic exposure modulates the virulence and antibiotic resistance of P. aeruginosa, raising concerns about the public health risks posed by the convergence of arsenic-contaminated water and multidrug-resistant bacteria.Copyright © 2025 Elsevier B.V. All rights reserved.