Objectives: The object of this study was to observe the in vitro antibacterial effects of Yeonkyokeumpae-jeon (YKKPJ) have been used for treating various gynecological diseases including mastitis in Korea, and individual six kinds of herbal composition aqueous extracts - Forsythiae Fructus (FF), Millettiae Caulis (MC), Lonicerae Flos (LF), Fritillaria Thunbergii Bulb (FT), Taraxci Herba (TH) and Prunellae Spica (PS) against E. coli. Methods: Antibacterial activities against E. coli of YKKPJ, FF, MC, LF, FT, TH and PS aqueous extracts were detected using standard agar microdilution methods. In addition, the effects on the bacterial growth curve were also monitored at MIC and $MIC{\times}2$ levels. The effects on the intracellular killing and bacterial invasion of individual test materials were also observed using Raw 264.7 and MCF-7. The results were compared with ciprofloxacin, a second generation of quinolone antibiotics in the present study. Results: MIC of YKKPJ, FF, MC, LF, FT, TH, PS aqueous extracts against E. coli were detected as $0.039{\pm}0.013mg/ml$, $0.064{\pm}0.033mg/ml$, $0.108{\pm}0.053mg/ml$, $0.078{\pm}0.027mg/ml$, $16.250{\pm}8.385mg/ml$, $15.625{\pm}9.375mg/ml$, $0.254{\pm}0.131mg/ml$, repectively. YKKPJ, FF, MC, LF, FT, TH, PS aqueous extracts showed antibacterial effects against to E. coli, except for FT and TH, which were showed negligible antibacterial effects, respectively. In addition, ciprofloxacin with YKKPJ, FF, MC, LF and PS aqueous extracts also showed marked dosage-dependent inhibition of bacterial growth, and favorable inhibitory effects on the both bacterial invasion and intracellular killing assays using MCF-7 and Raw 264.7 cells were detected in this experiment. Conclusions: The results obtained in this study suggest that traditional polyherbal formula YKKPJ aqueous extracts showed more favorable antibacterial activities as compared to individual six kinds of herbal composition aqueous extracts. The antibacterial effects of YKKPJ against E. coli considered as results of complicated synergic effects of their six kinds of herbal components rather than simple antibacterial effects of single herbal components. It means, YKKPJ aqueous extracts may show potent anti-infectious effects against E. coil for mastitis.