This study investigated the synergistic effects of lactic acid (0.5%, 1% and 2%) and ultrasound (400 W, 20 kHz) on the inactivation of Pseudomonas fluorescens. Combined ultrasound and 2% lactic acid for 5 min reduced the planktonic cell count below 1.40 log cfu/mL. Ultrasound promoted the inactivation of P. fluorescens biofilms by lactic acid and increased the release of ATP and nucleic acids from bacterial cells by 0.09–0.22 μmol/OD and 0.043–0.113 of OD260 values, respectively. Results indicated that combined ultrasound and lactic acid can serve as a potential technique to control P. fluorescens biofilms.