Background: Healthcare-associated infections (HAIs) represent an ongoing problem for all clinics. Children’s clinics have waiting rooms that include toys and activities to entertain children, possibly representing reservoirs for HAIs. This study focuses on a newly constructed children’s outpatient clinic associated with a teaching hospital. We studied waiting room bacterial colonization of floors and play devices from the last phase of construction through 6 months of clinical use. Methods: Waiting room areas on the first 2 floors of the facility were studied due to high patient volume in those areas. In total, 16 locations were sampled: 11 on floors and 5 on play items. Using sterile double-transport swabs, all locations were sampled on 5 separate occasions over 2 months during the last phase of construction and 13 times over 6 months after the clinic was opened. After collection swabs were placed on ice, transported to a microbiology lab, and used to inoculate Hardy Diagnostics Cdiff Banana Broth (for Clostridium difficile - Cdiff), CHROM MRSA agar (for methicillin resistant Staphylococcus aureus - MRSA), Pseudomonas isolation agar (for Pseudomonas spp and P. aeruginosa), and tryptic soy agar to detect Bacillus spp. Media were incubated for 48 hours at 37°C and were scored for bacterial presence based on observation of colonies or change in the medium. Results: During the construction phase, waiting-room-floor bacterial colonies were dominated by Bacillus spp, and first-floor waiting rooms had nearly 7 times more colonies than those on the second floor (P < .05). A similar pattern was observed for C. difficile and MRSA. No Pseudomonas spp were observed during construction. Once patients were present, Bacillus spp contamination dropped for the first floor, but increased for the second floor. All other bacterial types (C. difficile, MRSA, Pseudomonas spp, and P. aeruginosa) increased on the second floor after the clinic opened (eg, from 23% to 42% for C. difficile and from 7% to 46% for MRSA; P < .05). The play devices showed small increases in bacterial load after clinic opening, most notably Pseudomonas spp. Conclusions: This study provides evidence that a shift from bacterial species associated with soil (eg, Bacillus spp) toward species commonly associated with humans occurred in waiting rooms after construction in this children’s outpatient clinic. Increases for MRSA, Pseudomonas spp, and P. aeruginosa were linked to patient presence. These data suggest that patients, their families, and clinic staff transport bacteria into clinic waiting rooms. This outpatient clinic environmental contamination may increase potential for HAIs and may represent a target for intervention.