ABSTRACTAztreonam-avibactam, eravacycline, and cefoselis are three novel antimicrobial agents for the treatment of serious infections caused by Gram-negative bacteria. We evaluated thein vitroactivities of the above-mentioned three antimicrobial agents against clinicalEnterobacteralesisolates. A total of 1,202Enterobacteralesisolates, including 10 genera or species, were collected from 26 hospitals that cover seven regions of China. The susceptibilities of the 30 antimicrobial agents were interpreted based on the combination of U.S. Food and Drug Administration and Clinical and Laboratory Standards Institute guidelines. The results indicated that allEnterobacteralesisolates showed high susceptibility to aztreonam-avibactam (98.25%), eravacycline (85.69%), and cefoselis (62.73%). The first two antimicrobial agents also demonstrated potent activities against multidrug-resistant and carbapenem-resistantEnterobacteralesindependent of antimicrobial resistance mechanisms. The rates of susceptibility to aztreonam-avibactam, eravacycline, and cefoselis were lowest inMorganellaspp. (84.42%),Proteusspp. (33.65%), andEscherichia coli(40.14%), respectively. In general, the lower rates of susceptibility to eravacycline and cefoselis were in the older inpatient group. The strains isolated from urinary tract exhibited the lowest rate of susceptibility (78.97%) to eravacycline, and the lowest rate of susceptibility (45.83%) to cefoselis was observed in nervous system specimens. The strains isolated from intensive care unit (ICU) wards showed significantly reduced susceptibility to cefoselis compared with those isolated from non-ICU wards. The MIC values of aztreonam-avibactam and ceftazidime-avibactam have poor consistency (weighted kappa = 0.243), as did eravacycline and tigecycline (weighted kappa = 0.478). Cefoselis and cefepime showed highly similar activities againstEnterobacterales(weighted kappa = 0.801). Our results support the clinical development of aztreonam-avibactam, eravacycline, and cefoselis to treat infections caused byEnterobacterales.IMPORTANCEInfections caused by multidrug-resistant (MDR)Enterobacterales, especially carbapenem-resistantEnterobacterales(CRE), have been a challenging clinical problem due to the limited therapeutic options. Therefore, the need to develop novel antimicrobial agents and evaluate their activities againstEnterobacterales in vitrois urgent. Our results show that the novel antimicrobial agents aztreonam-avibactam and eravacycline retain activities against MDR and CRE isolates, including carbapenemase producers and non-carbapenemase producers. Further analysis combined with clinical information on the strains tested revealed that no significant differences were observed in susceptibility rates of strains with different demographic parameters to aztreonam-avibactam. Age, specimen source, and department were associated with the susceptibility of strains to eravacycline and cefoselis (P≤ 0.01). Compared with ceftazidime-avibactam, aztreonam-avibactam has its advantages and limitations againstEnterobacterales. The potent activity of eravacycline againstEnterobacteraleswas higher than that of tigecycline. Cefoselis and cefepime showed a highly consistent activity againstEnterobacterales.