Polysorbate 80 (PS80) is a nonionic surfactant and detergent that inhibits biofilm formation by Pseudomonas aeruginosa at concentrations as low as 0.001% and is well tolerated in human tissues. However, certain clinical and laboratory strains (PAO1) of P. aeruginosa are able to form biofilms in the presence of PS80. To better understand this resistance, we performed transposon mutagenesis with a PS80-resistant clinical isolate, PA738. This revealed that mutation of algC rendered PA738 sensitive to PS80 biofilm inhibition. AlgC contributes to the biosynthesis of the exopolysaccharides Psl and alginate, as well as lipopolysaccharide and rhamnolipid. Analysis of mutations downstream of AlgC in these biosynthetic pathways established that disruption of the psl operon was sufficient to render the PA738 and PAO1 strains sensitive to PS80-mediated biofilm inhibition. Increased levels of Psl production in the presence of arabinose in a strain with an arabinose-inducible psl promoter were correlated with increased biofilm formation in PS80. In P. aeruginosa strains MJK8 and ZK2870, known to produce both Pel and Psl, disruption of genes in the psl but not the pel operon conferred susceptibility to PS80-mediated biofilm inhibition. The laboratory strain PA14 does not produce Psl and does not form biofilms in PS80. However, when PA14 was transformed with a cosmid containing the psl operon, it formed biofilms in the presence of PS80. Taken together, these data suggest that production of the exopolysaccharide Psl by P. aeruginosa promotes resistance to the biofilm inhibitor PS80.