Enterobacter cloacae are thought to exhibit resistance to third-generation cephalosporins (3GCs) despite their sensitivity to these drugs; therefore, broad-spectrum antibiotics such as carbapenems are primarily used against them. Reports elucidating the treatment outcomes of 3GCs against E. cloacae are limited; therefore, we aimed to accumulate further evidence in this regard. Patients with isolated E. cloacae infections who were treated with antibiotics between April 2008 and April 2021 at Hokkaido University Hospital were included in the study. The primary endpoint was the difference in treatment efficacy between patients treated with the 3GCs after the detection of E. cloacae and those treated with other drugs. As a secondary endpoint, we compared the differences in treatment efficacy rates according to specimen type, severity, and type of antibiotics. Furthermore, multivariate analysis was performed to identify the factors influencing treatment failure. Among the 146 cases analyzed, 25 and 121 were categorized into the 3GCs and others groups, respectively. The treatment efficacy rate did not significantly differ between the two groups (3GC vs. others: 80.0% vs. 84.3%; P = 0.564). Only one patient in the 3GCs group developed resistance. The treatment efficacy rate did not differ according to the specimen type, severity, or antibiotic class between the groups. Multivariate analysis confirmed that the use of 3GCs did not affect treatment failure. 3GCs may be considered a potential therapeutic option for E. cloacae infections.