Air-based atmospheric-pressure plasma is an effective non-thermal method in deactivating various kinds of microbial biofilms with several advantages, including high bactericidal efficiency and low treatment costs. Bacterial biofilm formation is a major determinant in establishment of bacterial infection and also resistance to antibacterial chemotherapy. This study aims to assess the anti-biofilm potential of air-based atmospheric-pressure DBD plasma against Staphylococcus aureus and Escherichia coli biofilms. The biofilms of Staphylococcus aureus and Escherichia coli were exposed to air-based atmospheric-pressure DBD plasma for up to 4 min (control, 30 s, 90 s, 3 min, and 4 min) and their biofilm formation level, viability, and membrane integrity were determined. Based on the results, plasma exposure caused disruption up to 70% and 85% for S. aureus and E. coli biofilms, respectively. The biofilm disruption potential of air-based atmospheric-pressure DBD plasma was confirmed using the scanning electron microscopy (SEM). Besides, based on confocal laser scanning microscopy (CLSM), plasma exposure caused a significant bacterial inactivation and E. coli was found as more susceptible strain than S. aureus. In conclusion, atmospheric-pressure DBD plasma could be considered an efficient non-thermal approach against bacterial pathogenicity by biofilm disruption and thus prevention of infection establishment.