The preparation of Enterococcus faecalis RSI is used as a therapeutics for human intestinal disorders. However, the microbe in this preparation is usually very sensitive to rifampicin and fluoroquinolones. If this preparation is taken with rifampicin or fluoroquinolones, its therapeutic effect can not be expected. E. faecalis RFR11, containing resistance to rifampicin was obtained by MNNG mutation method. Serial passage of E. faecalis RFR11 produced E. feacalis OFR16 on agar with 2-fold minimal inhibitory concentration of ofloxacin produced. E. feacalis OFR16 was resistant to fluoroquinolones up to 8-256 fold higher than that for the original strain. E. faecalis OFR16 also exhibited identical characteristics with the parent strain when they were tested for lactic acid formation and growth inhibition of E. coli MB4-5737 and Shigella sonnei MB4-10411. From in vitro test, it was identified that rifampicin and ofloxacin is not inactivated by certain factors of E. faecalis OFR16. Conclusively. E. faecalis OFR16, rifampicin and fluoroquinolones resistant mutant, is an efficient strain that has insensitivity against rifampicin and fluoroquinolones and original biochemical characteristics of the parent strain.