Objective: The rapid and accurate detection of bacterial infections and antibiotic susceptibility is crucial for initiating appropriate treatment. This study aimed to compare the antimicrobial resistance of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa strains using traditional disk diffusion and rapid antibiotic susceptibility testing in our hospital’s medical microbiology laboratory. Methods: The study included 51 E. coli, 26 S. aureus and 25 P. aeruginosa isolates including the standard strain. All isolates were tested by Kirby-Bauer disc diffusion method in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 guidelines. AST FAST ES and AST FAST NF media (Diagnotis A.Ş., Turkey) were used for rapid antibiotic susceptibility testing and evaluated separately in accordance with the manufacturer’s instructions and EUCAST’s breakpoint table. Results: In E. coli isolates, very major error (VME) were found 1.96% for most of antibiotics and 3.92% for cefepime. No VME detected for amoxicillin-clavulanic acid, ciprofloxacin, and meropenem. The major error (ME) rates were 5.88% for ampicillin, 3.9% for cefuroxime and ceftazidime, and 1.96% for meropenem. The categorical agreement of E. coli strains was above 90% for all antibiotics. In the S. aureus isolates, VME was found in 3.85% of strains for clindamycin, gentamicin, and moxifloxacin and 15.38% for erythromycin and cefoxitin. VME did not detect of fusidic acid, trimethoprim-sulfamethoxazole (SXT), mupirocin, linezolid, and amikacin. A major error was 19.23% for SXT, 11.53% for cefoxitin, and 3.85% for moxifloxacin. The categorical agreement was above 90% for several antibiotics but below acceptable levels for cefoxitin (73.08%), SXT (80.77%), and erythromycin (84.61%). In P. aeruginosa strains, VME rates were determined to be as 11.5% for meropenem and 3.9% for amikacin. Major errors were not detected. The categorical agreement was found to be above the acceptance criteria for amikacin-aztreonam and imipenem. Conclusion: Our study showed that the rapid antibiotic susceptibility test can be used for early detection of E. coli susceptibility results in medical microbiology laboratories. However, standard methods should confirm results due to high ESRD rates. On the other hand, the data obtained for P. aeruginosa and S. aureus should be supported with more samples and that studies to standardize the rapid antibiotic susceptibility test medium should be continued.