•CO2-assisted inactivation of Escherichia coliand Staphylococcus aureussubject to HPP in liquid samples was observed.•CO2-HPP made up the deficiency of HPP on Gram-positive bacteria.•As compared with HPP, CO2-HPP showed more severe damage on morphology and intracellular structure of E. coliand S. aureus.•The penetration, explosion, and acidification effects of CO2has played on E. coliand S. aureusby HPP.