Because of the high incidence ofPseudomonas aeruginosabiofilms-related nosocomial infections, venoms from common Thai snakes were tested. Although venoms from king cobra (Ophiophagus hannah; OH) and green pit viper (Trimeresurus albolabris) showed the broadest antibacterial spectrum, OH venom demonstrated more profound anti-biofilm activities againstP. aeruginosa. Additionally, purifiedl-amino acid oxidase from OH venom (OH-LAAO), using a three-step chromatography and protein identification, reduced biofilm mass as indicated by the downregulation of several genes, including the genes for biofilm synthesis (algDandpslB) and biofilm regulators (algU,gacA, andsiaD). Moreover, OH-LAAO disruptedPseudomonas-preformed biofilms via upregulation of several genes for biofilm dispersion (nbdA,bdlA, anddipA) and biofilm degradation (endAandpslG), resulting in a reduction of the biofilm biomass. Due to the antimicrobial effects and anti-biofilm activities (reduced production plus increased dispersion) neutralized by catalase, a hydrogen peroxide (H2O2)-degrading enzyme, the enhanced H2O2by OH venom might be one of the anti-biofilm mechanisms. Hence, OH-LAAO was proposed as a novel agent againstPseudomonasbiofilms for either treatment or prevention. More studies are interesting.