Bacterial resistance is a global problem with serious consequences: increased mortality, prolonged hospitalizations, higher rates of surgical site infection, and increased costs. We monitor resistance profiles for epidemiological assessment, guidance of targeted antimicrobial therapies, and identification of new strains. Rapid in vitro multiplex immunoassays exist for phenotypic detection and differentiation of five carbapenemase families (KPC, OXA-48-like, VIM, IMP, and NDM) directly from bacterial colonies. NG-Test Carba 5 is a rapid in vitro multiplex immunoassay (≤ 15 min) for phenotypic detection and differentiation of these five common carbapenemase families produced by Enterobacteriales and Pseudomonas. Tests were performed in the laboratory from August 2024 to March 2025. Sixty strains were tested (49 Enterobacteriales and 11 Pseudomonas aeruginosa) resistant to carbapenems identified by Vitek MS Prime (Biomerieux), selecting the carbapenem-resistant bacteria. NG-Test Carba 5 was used to identify the enzyme responsible for the resistance mechanism. We found 60 carbapenem-resistant bacteria, 49 (81.67%) Enterobacteriales, of which 45 (75%) were Klebsiella pneumoniae (88.89% KPC producers, 2.22% NDM metallo-β-lactamase, and 8.88% with no enzyme identified) and 4 (6.67%) other Enterobacteriales: 1 (25%) Klebsiella cloacae producing NDM metallo-β-lactamase, 2 (50%) Escherichia coli producing KPC, and 1 (25%) Proteus mirabilis with no enzyme identified. KPC production is the main isolated enzymatic mechanism among enterobacteria, but other enzymes such as metallo-β-lactamases are emerging. Eleven (18.33%) Pseudomonas aeruginosa were isolated: 1 (9.10%) KPC producer, 2 (18.18%) VIM producers, and 8 (72.72%) negative tests. A multicenter study conducted between 2018 and 2019 showed that KPC and VIM are the main enzymes isolated in Pseudomonas. In 13 (21.67%) bacteria the test was negative, either because they did not produce enzymes targeted by the test or because they produced other resistance mechanisms. These tests allow monitoring the emergence of new enzymes that confer antimicrobial resistance, enabling preventive measures and therapeutic adjustments that improve cost-effectiveness in clinical practice.