The aim of this study was to investigate the inactivation effect of organic acids and ultrasound against Escherichia coli and Listeria monocytogenes biofilms on the surface of lettuce leaves and to determine the effect of these inactivation treatments on the quality characteristics of the lettuce. Lettuce leaves with biofilms formed following inoculation with E. coli and L. monocytogenes were treated individually or simultaneously with ultrasound and different organic acids (acetic acid, malic acid, citric acid and lactic acid) for periods of 10, 30 and 60 min. Combined treatments caused synergistic and antagonistic inactivation of pathogen biofilms. In addition, the anti-biofilm effect of combined treatments was found to be higher compared with a single treatment. With an increase in treatment time from 10 min to 60 min, biofilm detachment rate was increased on the surface of the lettuce. The reduction in E. coli and L. monocytogenes biofilms varied from 0.43 to 5.54 log CFU/cm2 and from 0.40 to 4.23 log CFU/cm2, respectively. Lactic acid and malic acid showed stronger anti-biofilm activities than the other organic acids. All these results indicate that biofilm inactivation on lettuce samples depends on target microbial species, the type of organic acid, the treatment time, and method. Additionally, decontamination treatments for 60 min did not adversely affect the colour, texture or sensory quality of the lettuce. In conclusion, the combined treatment of ultrasound and organic acid is suggested as a promising hurdle technology in order to remove pathogen biofilms from fresh produce.