To evaluate the in vitro antimicrobial activities of biapenem, arbekacin, and cefminox against different gram-negative bacterial isolates in China, a total of 100 non-duplicated Escherichia coli, 100 Acinetobacter baumannii, 100 Pseudomonas aeruginosa, and 99 Klebsiella pneumoniae isolates were collected from 6 teaching hospitals in China in 2012. The minimal inhibitory concentrations (MICs) of biapenem, arbekacin, cefminox and 13 other antibiotics were determined by the broth microdilution method. The carbapenems (biapenem, meropenem, and imipenem) exhibited high antimicrobial activity against E. coli (98%) and K. pneumoniae (≥95%), followed by colistin and amikacin. The MIC50 and MIC90 of biapenem against E. coli were ≤0.06 mg/L and 0.25 mg/L, respectively. For K. pneumoniae, the MIC50 and MIC90 of biapenem were 0.25 mg/L and 1.0 mg/L, respectively. The MIC50 and MIC90 of cefminox against E. coli were 1.0 mg/L and 4.0 mg/L, respectively. The resistance rates of A. baumannii to most of the antibiotics were more than 50%, except for colistin. Amikacin was the most active antibiotic against P. aeruginosa (97%), followed by colistin (93%). The MIC50 and MIC90 of arbekacin against P. aeruginosa were 2.0 mg/L and 8.0 mg/L, respectively. In conclusion, carbapenems, colistin, amikacin, and arbekacin exhibited high antimicrobial activities against gram-negative bacteria, except A. baumannii.