infection (CDI) is most commonly diagnosed using nucleic acid amplification tests (NAAT); the low positive predictive value of these assays results in patients colonized with unnecessarily receiving CDI treatment antibiotics. The risks and benefits of antibiotic treatment in individuals with such cases are unknown. Fecal samples of NAAT-positive, toxin enzyme immunoassay (EIA)-negative patients were collected before, during, and after randomization to vancomycin ( = 8) or placebo ( = 7). and antibiotic-resistant organisms (AROs) were selectively cultured from fecal and environmental samples. Shotgun metagenomics and comparative isolate genomics were used to understand the impact of oral vancomycin on the microbiome and environmental contamination. Overall, 80% of placebo patients and 71% of vancomycin patients were colonized with posttreatment. One person randomized to placebo subsequently received treatment for CDI. In the vancomycin-treated group, beta-diversity (0.0059) and macrolide-lincosamide-streptogramin (MLS) resistance genes (0.037) increased after treatment; and vancomycin-resistant enterococci (VRE) environmental contamination was found in 53% of patients and 26% of patients, respectively. We found that vancomycin alters the gut microbiota, does not permanently clear , and is associated with VRE colonization/environmental contamination. (This study has been registered at ClinicalTrials.gov under registration no. NCT03388268.) A gold standard diagnostic for infection (CDI) does not exist. An area of controversy is how to manage patients whose stool tests positive by nucleic acid amplification tests but negative by toxin enzyme immunoassay. Existing data suggest most of these patients do not have CDI, but most are treated with oral vancomycin. Potential benefits to treatment include a decreased risk for adverse outcomes if the patient does have CDI and the potential to decrease shedding/transmission. However, oral vancomycin perturbs the intestinal microbiota and promotes antibiotic-resistant organism colonization/transmission. We conducted a double-blinded randomized controlled trial to assess the risk-benefit of oral vancomycin treatment in this population. Oral vancomycin did not result in long-term clearance of , perturbed the microbiota, and was associated with colonization/shedding of vancomycin-resistant enterococci. This work underscores the need to better understand this population of patients in the context of /ARO-related outcomes and transmission.