Polyphenols exhibit potential functional activities, especially rosmarinic acid (RosA) and caffeic acid (CafA). In this study, two different methods, ultrasonic-assisted ethanol extraction (60%) and ultrasound-assisted cellulase (≥15 000 Ug-1, 2%) hydrolysis, were used for the extraction of the total phenolics from 44 species of Perilla frutescens. The Folin-Ciocalteu method of detection showed that the content of the total phenolics extracted by cellulase hydrolysis was the highest and attained up to 28.00 mgGAE per gextracts for ZB1. Continuously, the extracts were purified using XDA-8 macroporous resin and medium-pressure liquid chromatography (MPLC), and the content of the total phenolics improved to 66.62 mgGAE per gextract. A high-performance liquid chromatography (HPLC) assay showed that the total polyphenols were mainly composed of gallic acid, caffeic acid, rosmarinic acid, luteolin and apigenin. Besides, a sequential XDA-8 macroporous resin combined with high-speed counter-current chromatography (HSCCC)/MPLC system was established for the simultaneous isolation and preparation of RosA (purity 98.29%) and CafA (purity 97.01%) from the extracts. Furthermore, the antibacterial activities of the total polyphenols were evaluated by the disc diffusion method and scanning electron microscopy (SEM) observation. The results verified that the total polyphenols had effective antibacterial activity on three kinds of bacteria including E. coli, S. aureus, and B. subtilis in a concentration-dependent manner. All of these results demonstrated that the ultrasound-assisted cellulase hydrolysis extraction of the total polyphenols and the proposed three-step separation of RosA and CafA gave high yields and good purity, and they exhibited effective antibacterial ability.