Pseudomonas aeruginosa is the most common Gram-negative bacteria that causes fatal nosocomial infections amongst hospitalized patients, especially immunocompromised subjects. The bacteria resist a wide variety of currently available antibiotics. One of the resistant mechanisms includes forming biofilms in the infected host, which prevents the accessibility of drugs to their respective bacterial cell targets. A novel strategy to overcome this antibiotic resistance mechanism is the use of antibiotics in combination with biofilm formation inhibitors. The objective of this study was to investigate the effect of mulberry-leaf extracts on P. aeruginosa biofilm formation. The results of specific biofilm formation (SBF) index evaluations showed that mulberry-leaf ethanolic (256 g/ml) and ether extracts (32 g/ml) presented strong capacities (SBF index > 1.1) to reduce biofilm formation by 44.0% and 44.8%, respectively. Each plant extract anti-biofilm activity was tested further in combination with gentamicin (0.125 to 1,024 µg/mL) using the checkerboard method; however, no synergistic efficacy was observed. The MBEC and CLSM results revealed that the ether extract (32 g/ml) was effective against P. aeruginosa biofilm formation. This study should provide an alternative strategy for the effective treatment of deadly infections caused by the most common drug resistant bacteria, P. aeruginosa.