The most common clinical manifestation of Vibrio cholerae non-O1 non-O139 is gastroenteritis. This vibrion may also cause bacteremia, soft tissue infection, and other extraintestinal invasive disease, especially in immunocompromised patients. This study evaluated the current status of antimicrobial resistance in clinical isolates of V. cholerae non-O1 non-O139 in Taiwan as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. Minimal inhibitory concentrations (MICs) of 9 antimicrobial agents were determined by the agar dilution method. All of the isolates were susceptible to minocycline (MIC at which 90% of the isolates were inhibited [MIC(90)], 0.12 microg/mL), cefotaxime (MIC(90), 0.06 microg/mL), lomefloxacin (MIC(90), 0.12 microg/mL), levofloxacin (MIC(90), 0.03 microg/mL), ciprofloxacin (MIC(90), 0.03 microg/mL), moxifloxacin (MIC(90), 0.06 microg/mL), sparfloxacin (MIC(90), 0.06 microg/mL), gatifloxacin (MIC(90), 0.03 microg/mL), and cefazolin (MIC(90), 8 microg/mL). We conducted time-kill studies to evaluate the inhibitory activities of either cefazolin or minocycline alone or in combination against V. cholerae non-O1 non-O139 (Vc2). We also evaluated the inhibitory activity of cefazolin or cefotaxime combined with minocycline. The individual MICs of cefazolin, cefotaxime, and minocycline were 4 microg/mL, 0.0075 microg/mL, and 0.12 microg/mL, respectively, when approximately 5 x 105 colony-forming units/mL of V. cholerae non-O1 non-O139 was incubated. Bacterial growth was inhibited initially but resumed later when cefazolin, cefotaxime, or minocycline was used alone. When cefazoline or cefotaxime was combined with minocycline, V. cholerae non-O1 non-O139 was inhibited over 48 h and no regrowth was noted. We conclude that the combination of cefazolin or cefotaxime with minocycline has a synergistic inhibitory effect on V. cholerae non-O1 non-O139 in vitro.