Bacteria, when adhered to a substratum, can form biofilms. Nevertheless, many factors dictate biofilm formation and virulence factor production, including a response by the bacteria to their surroundings. This system is referred to as Quorum sensing (QS) also known as cell-cell communication. Pseudomonas aeruginosa is an infection causing agent in immune-compromised patients, it uses acyl-homoserine lactone (AHL) to coordinate its QS systems. In this work, the connection between some members of AHL produced by P. aeruginosa PAO1 and its biofilm development and the production of virulence factor was investigated. It was discovered that N-butanoyl-homoserine lactone (C4-HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL) perform a more consequential and eminent function in the biofilm maturation and virulence factor production while N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) plays a role in biofilm initiation. Because QS has been reported to be required for biofilm development and pathogenesis of P. aeruginosa, the results of this work have great importance and significance for the design of strategies for the control and prevention of biofilms.