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Biofilm acts as a complex barrier against antibiotics. In this study, we investigated the inhibitory activities of <i>Olea europaea</i> (olive) leaves <i>Camellia sinensis</i> (green tea), <i>Styrax benzoin</i>, <i>Ocimum basilicum</i>, <i>Humulus lupulus</i>, <i>Ruta graveolens</i>, and Propolis extracts on the biofilm formation, pyocyanin production, and twitching motility of <i>Pseudomonas aeruginosa</i> isolates. Moreover, we investigated the effect of olive leaf extract on the transcription of some biofilm related genes. A total of 204 isolates of <i>Pseudomonas</i> were collected from different Egyptian hospitals. A susceptibility test, carried out using the disc diffusion method, revealed that 49% of the isolates were multidrug-resistant. More than 90% of the isolates were biofilm-forming, of which 26% were strong biofilm producers. At subinhibitory concentrations, green tea and olive leaf extracts had the highest biofilm inhibitory effects with 84.8% and 82.2%, respectively. The expression levels of <i>lasI</i>, <i>lasR</i>, <i>rhlI</i>, and <i>rhlR</i> treated with these extracts were significantly reduced (<i>p</i> < 0.05) by around 97–99% compared to untreated isolates. This study suggests the ability of olive leaf extract to reduce the biofilm formation and virulence factor production of <i>P. aeruginosa</i> through the down regulation of quorum sensing (QS) genes. This may help in reducing our dependence on antibiotics and to handle biofilm-related infections of opportunistic pathogens more efficiently.