Background: Rapid screening of patients for colonization with carbapenemase-producing organisms (CPO), coupled with implementation of infection prevention strategies, has the potential to contain the spread of CPO. Methods: We first evaluated the performance of Xpert Carba-R assay (in comparison with other phenotypic methods) for carbapenemase detection using clinical isolates, and then used it to determine the intestinal CPO colonization in hospitalized patients. We then assessed the effectiveness of patient isolation in controlling the spread of CPO in a medical intensive care unit. Results: The Xpert Carba-R assay required the least processing time to reveal results and showed a 94.5% sensitivity and specificity in carbapenemase detection, except for IMP-8 (n = 4). During a 6-month study period, 134 patients in one ward were studied for CPO colonization and infection. Fifteen patients (11.2%) were colonized by CPO as detected by Xpert Carba-R assay, including three NDM, three IMP, and nine KPC possessing strains. The overall colonization and CPO infection rates were both 11.2% each. Isolation of patients with CPO led to a reduction in both colonization (from 28.6 to 5.6%) and infection rates (from 35.7 to 2.8%) during the study period (p < 0.05). Conclusion: Active surveillance of CPO utilizing the Xpert Carba-R assay supplemented with immediate patient isolation, proved to be an effective strategy to limit the spread of CPO in a health care setting.