To assess potential transmission of antibiotic-resistant organisms (AROs) using surrogate markers and bacterial cultures.Design: Pilot study.Setting: A 1,260-bed tertiary-care academic medical center.Participants: The study included 25 patients (17 of whom were on contact precautions for AROs) and 77 healthcare personnel (HCP).Methods: Fluorescent powder (FP) and MS2 bacteriophage were applied in patient rooms. HCP visits to each room were observed for 2-4 hours; hand hygiene (HH) compliance was recorded. Surfaces inside and outside the room and HCP skin and clothing were assessed for fluorescence, and swabs were collected for MS2 detection by polymerase chain reaction (PCR) and selective bacterial cultures.Results: Transfer of FP was observed for 20 rooms (80%) and 26 HCP (34%). Transfer of MS2 was detected for 10 rooms (40%) and 15 HCP (19%). Bacterial cultures were positive for 1 room and 8 HCP (10%). Interactions with patients on contact precautions resulted in fewer FP detections than interactions with patients not on precautions (P <.001); MS2 detections did not differ by patient isolation status. Fluorescent powder detections did not differ by HCP type, but MS2 was recovered more frequently from physicians than from nurses (P =.03). Overall, HH compliance was better among HCP caring for patients on contact precautions than among HCP caring for patients not on precautions (P =.003), among nurses than among other nonphysician HCP at room entry (P =.002), and among nurses than among physicians at room exit (P =.03). Moreover, HCP who performed HH prior to assessment had fewer fluorescence detections (P =.008).Conclusions: Contact precautions were associated with greater HCP HH compliance and reduced detection of FP and MS2.