Antimicrobial stewardship programs promote optimal antimicrobial use in the individual patient and reductions in inappropriate antimicrobial use in health care to limit the selective pressures that contribute to the development and spread of multidrug-resistant organisms. These efforts presume that the antimicrobial susceptibility testing (AST) data provided by the microbiology laboratory, which serve as the basis for clinical decision making, are accurate and delivered in a timely fashion. However, routine AST data are often not available until two to three days after the clinical specimen for culture has been collected from the patient. Nucleic acid amplification methods can provide data on the presence of antimicrobial-resistant microorganisms directly from clinical specimens often in less than 1 hour. Organisms causing sepsis, respiratory tract infections, and sexually transmitted diseases are all amenable to rapid detection. Several commercial molecular diagnostic assays are already in use in microbiology laboratories worldwide; however, physician acceptance of the rapid methods is often slow. Data on the presence of resistant bacteria in a clinical sample in less than one hour should improve the effectiveness of antimicrobial stewardship programs.