Bacteria use different types of motilities in order to colonize and adapt to new environments. These motilities also play an important role in the formation of biofilm, allowing bacteria to develop resistance to antibiotics and host’s immune systems. The objective of this study was to evaluate the ability of essential oils of Lippia multiflora flowers to inhibit biofilm formation and motility in Pseudomonas aeruginosa PAO1. The capacity of essential oil of Lippia multiflora flowers to inhibit biofilm formation in Pseudomonas aeruginosa was evaluated spectrophotometrically by using the crystal violet method. The property of the essential oil to inhibit different types of motilities such as swimming, swarming and twitching was further evaluated by measuring the diameters of bacterial migration on liquid or semiliquid Luria Bertani agar medium. The essential oil exhibited good anti-biofilm activity in Pseudomonas aeruginosa. At a concentration of 1%, essential oil presented a degree of biofilm inhibition similar to that of salicylic acid used as a reference (P > 0.05). In addition, the essential oil significantly inhibited swarming, twitching and swimming in Pseudomonas aeruginosa compared to the control (P < 0.001). The inhibition of biofilm formation as well as that of bacterial motility increases with the concentration of the essential oil. The essential oil of Lippia multiflora flowers possesses an anti-bacterial potential to fight against multi-resistant bacteria.