Objective Automated systems are needed for the rapid and accurate diagnosis of Pseudomonas-associated nosocomial infections among critically ill patients admitted to the intensive care unit. We assessed the performance of TDR-300B and VITEK®2 for the identification of P. aeruginosa using VITEK®-MS as the gold standard. Methods This analytical study employed a cross-sectional approach. First, 44 clinical isolates of P. aeruginosa were collected and refreshed. Next, a single colony of oxidase-positive, gram-negative rods (30 samples) was inoculated into a TDR-300B NF-64 card and VITEK®2 GN cassette for each isolate. Finally, bacterial identification was performed using VITEK®-MS for comparative analysis. Results Compared with the results for VITEK®-MS, the congruence rates for TDR-300B and VITEK®2 were 80.76% (21/26) and 92.30% (24/26), respectively. Further, high sensitivity was observed for TDR-300B and VITEK®2 (95.45% and 100%, respectively). In addition, TDR-300B had a lower positive predictive value and accuracy than VITEK®2, albeit without significance. Conclusions Conclusively, there were no significant differences regarding the diagnostic efficiency of TDR-300B and VITEK®2 for P. aeruginosa.