To determine the efficacy of trovafloxacin as a possible treatment for intra-abdominal abscesses, we have developed an anaerobic time-kill technique using different inocula to study the in vitro killing of Bacteroides fragilis in pure culture or in mixed culture with either Escherichia coli or a vancomycin-resistant strain of Enterococcus faecium (VREF). With inocula of 5 × 10⁵ CFU/ml and trovafloxacin concentrations of 2 µg/ml, a maximum observed effect (E_max) of 6.1 (log₁₀ CFU/ml) was attained with all pure and mixed cultures within 24 h. With inocula of 10⁸ CFU/ml, a similar E_max and a similar concentration to produce 50% of E_max (EC₅₀) for B. fragilis were found in both pure cultures and mixed cultures with E. coli. However, to produce a similar killing of B. fragilis in the mixed cultures with VREF, a 14-fold increase in the concentration of trovafloxacin was required. A vancomycin-susceptible strain of E. faecium and a trovafloxacin-resistant strain of E. coli were also found to confer a similar "protective" effect on B. fragilis against the activity of trovafloxacin. Using inocula of 10⁹ CFU/ml, the activity of trovafloxacin was retained for E. coli and B. fragilis and was negligible against VREF. We conclude that this is a useful technique to study the anaerobic killing of mixed cultures in vitro and may be of value in predicting the killing of mixed infections in vivo. The importance of using mixed cultures and not pure cultures is clearly shown by the difference in the killing of B. fragilis in the mixed cultures tested. Trovafloxacin will probably be ineffective in the treatment of infections involving large numbers of enterococci. However, due to its ability to retain activity against large cultures of B. fragilis and E. coli, trovafloxacin could be beneficial in the treatment of intra-abdominal abscesses.