Aims The assessment of the bacterial viability of chlorine- and quaternary ammonium compounds (QACs)-treated Lactobacillus cells by culture-dependent and -independent methods. Methods and Results Lactobacillus isolates (Lactobacillus plantarum G1, Lactobacillus plantarum B1, Lactobacillus brevis S1 and Lactobacillus paracasei W1) in biofilm and planktonic cell suspensions were treated with chlorine-based (0 center dot 018 and 0 center dot 18%) and QACs-based (0 center dot 2 and 2 center dot 0%) disinfectants for 5 min and then analysed by plate counting, flow cytometry (FCM) and fluorescence activated cell sorting (FACS). The reaction of sessile cells to disinfectants was assessed with the confocal laser scanning microscopy (CLSM). Plate counts revealed L. paracasei W1 to be substantially inactivated by both disinfectants, while counts of the other isolates to be significantly reduced only by QACs, with L. plantarum B1 and L. brevis S1 showing a greater difference between QACs concentrations and cell types. In several cases, the disinfectants caused slightly higher inactivation of planktonic than biofilm cells, with L. plantarum B1 being significantly less sensitive to QACs in biofilm cells (P < 0 center dot 05). Following FCM with a SytoA (R) 9/PI assay, which addresses cell membrane integrity, the emergence of damaged (SytoA (R) 9(-)PI(+)) and injured (SytoA (R) 9(+)PI(+)) subpopulations was often observed in cells when they were treated with QACs, whereas intact (SytoA (R) 9(+)PI(-)) and unstained (SytoA (R) 9(-)PI(-)) subpopulations were mostly encountered in chlorine-treated cells. Except SytoA (R) 9(-)PI(+), all subpopulations were recovered on agar plates following FACS, with biofilm cells showing higher culturability irrespective of conditions, probably because of the residues of the biofilm matrix which serve as a protective cover for the bacteria. The CLSM revealed a substantial cell membrane damage within the QACs-treated biofilms, however, some cells deep in the biofilm were still intact and thus remained protected against this disinfectant. Conclusion We found that FCM/FACS proved useful in the analysis of lactobacilli membrane integrity in disinfection experiments as well as in recovery evaluation of planktonic-biofilm cell subpopulations. In turn, CLSM was particularly useful in investigating the resistance mechanism when Lactobacillus cells were embedded in biofilms. Significance and Impact of the Study This study highlights the need for treatment optimization on a case-by-case basis to avoid the emergence of cells in intermediate states with recovery potential and to reach and, thus, kill all bacteria in already developed lactobacilli biofilms.