The aim of the present study was to assess the in vitro antimicrobial activities of nanoliposomal formulations loaded with vancomycin or/and rifampin against the biofilm formed by Staphylococcus epidermidis at 37 °C under aerobic condition. Liposomal formulations were prepared by dehydration-rehydration (DRV) method and characterized for size, zeta potential and encapsulation efficacy. The ability of different formulations on eradication of bacterial biofilm was assessed through optical density ratio (ODr) and the results implicate higher survival rates of S. epidermidis on biofilm. Positive control was defined as an ODr = 1.0. The zeta potential of anionic, cationic and PEGylated liposomes was -35 ± 2, 35 ± 1 and 27 ± 2 mV whereas the mean sizes of these liposomal formulations were 145 ± 4, 134 ± 1 and 142 ± 6 nm, respectively. Encapsulation efficacy of rifampin and vancomycin was more than 60% and about 25%, respectively. Cationic liposomal rifampin lowered the ODr to 0.61 and was the most effective formulations against S. epidermidis biofilm (p<0.001). The antibiofilm activity of liposomal formulations was concentration- and time-dependent manner. The results of this study showed that rifampin-loaded liposomes were effective against bacterial biofilm.