This study was designed to evaluate the inactivation, detachment, and recovery properties of Listeria monocytogenes biofilms treated by the combined high hydrostatic pressure (HHP) with nisin under acidic conditions. Biofilm cells were treated at 400 MPa for 20 min in different trypticase soy broth (TSB) solutions, including TSB at pH 7.0 without nisin (TSB7-N), TSB at pH 7.0 with nisin (TSB7+N; 1,000 IU/mL), TSB at pH 5.0 without nisin (TSB5-N), and TSB at pH 5.0 with nisin (TSB5+N). The biofilm cells in TSB7+N were significantly inactivated after HHP treatment. The numbers of biofilm cells were reduced by more than 6 log CFU/mL in TSB5-N and TSB5+N. More noticeable changes in cell size and shape were observed in TSB5-N and TSB5+N, showing shortened and shrunk biofilm cells. These results demonstrate that the HHP treatment combined with nisin and acid could effectively inactivate L. monocytogenes biofilm and detached cells.