Hospital wastewater represents a unique type of waste that is extremely harmful to the general public it contains a wide variety of microbial species and related genomes. This work aimed to characterize the production of biofilm in hospital wastewater isolates of Pseudomonas aeruginosa at the genotypic and phenotypic levels. The strains were identified with cultural and biochemical characteristics, microscopic patterns, and the Vitek 2 System. The isolates were assessed for the measurement of the adhesion of cells to the microtiter plates. Polymerase Chain Reaction was used to determine quorum sensing (QS) genes. The current findings indicated that of the 70 P. aeruginosa isolates, 78.57% (55/70) were biofilm producers: 27 (47.27%) were classified as weak producers, and the rates of moderate and strong producers were 19 (34.54%) and 11 (20%), respectively. Furthermore. All examined isolates positively produced the QS genes. According to the findings, biofilm served as a survival mechanism for bacteria in hospital wastewater, providing bacteria with more favorable habitats than planktonic forms. It also implied the significance of QS in the production and persistence of P. aeruginosa biofilms by allowing the density of populations to be recognized. Therefore, the detection of these genes and their correlation with the production of biofilm may be useful in restricting the spread of pathogenic bacteria in wastewater.