Natural biologically active substances have received continuous attention for the potentially beneficial health properties against chronic diseases. In this study, bacteriostatic active substance from Camellia oleifera meal, which is a major byproduct of the Camellia oil processing industry, were extracted with continuous phase change extraction (CPCE) method and separated by HSCCC. Compared with traditional extraction methods, CPCE possessed higher extraction efficiency. Two main substances were separated and purified (above 90.0%). The structure of them were further identified by UV, LC-ESI-MS-MS, 1H-NMR, and 13C-NMR as flavonoids F2 kaempferol 3-O-[β-d-glucopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-dglucopyranoside and J2 kaempferol 3-O-[β-d-xylopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside for the first time in C. Oleifera meal. The results of antibacterial activity measurement showed that both compounds have excellent antibacterial activity. And the antibacterial stability of F2 were finally confirmed: F2 showed broad spectrum antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella enteriditis, Bacillus thuringiensis, Aspergillus niger and Rhizopus nigricans. Besides, F2 exhibited relatively high stable property even at high temperature, acid and metal ion solutions. The findings of this work suggest the possibility of employing C. oleifera meal as an attractive source of health-promoting compounds, and at the same time facilitate its high-value reuse and reduction of environmental burden.