Screening plant extracts for discovering new compounds effective in treating bacterial infections has been of great interest to scientists. Phytochemical plant extracts have shown direct antibacterial activity by affecting bacterial growth and metabolism. An EPS (extracellular polymeric substance) matrix protects a biofilm, a structural form of a microbial group. Biofilms are believed to be the major cause of infections and play an essential role in the development of drug resistance. In this study, we investigated the phytochemical composition and anti-biofilm properties of papaya (Carica papaya L.) leaf extract. The preliminary results of our studies indicate that papaya leaf extract contains a variety of bioactive compounds, including triterpenes, steroids, coumarins, quinones, and tannins. These extracts were able to prevent biofilm formation in Serratia marcescens, Pseudomonas aeruginosa, and Vibrio cholera. This study suggests that papaya leaf extracts can be used to develop anti-biofilm agents to control infections caused by these pathogens and prevent the formation of biofilms on medical devices.