There is limited data on the effects of discontinuing single-room isolation while maintaining contact precautions, such as the use of gowns and gloves. In April 2021, our hospital ceased single-room isolation for patients with VRE because of single-room unavailability. This study assessed the impact of this policy by examining the incidence of hospital-acquired VRE bloodstream infections (HA-VRE BSI). This retrospective quasi-experimental study was conducted at a tertiary care hospital in Seoul, South Korea. Time-series analysis was used to evaluate HA-VRE BSI incidence at the hospital level and in the hematology unit before (phase 1) and after (phase 2) the policy change. At the hospital level, HA-VRE BSI incidence level (VRE BSI per 1000 patient-days per month) and trend did not change significantly between phase 1 and phase 2 (coefficient -0.015, 95% confidence interval [CI]: -0.053 - 0.023, P = 0.45 and 0.000, 95% CI: -0.002 - 0.002, P = 0.84, respectively). Similarly, HA-VRE BSI incidence level and trend in the hematology unit (-0.285, 95% CI: -0.618 - 0.048, P = 0.09 and -0.018, 95% CI: -0.036 - 0.000, P = 0.054, respectively) did not change significantly across the two phases. Discontinuing single-room isolation of VRE-colonized or infected patients was not associated with an increase in the incidence of VRE BSI at the hospital level or among high-risk patients in the hematology unit. Horizontal intervention for multidrug-resistant organisms, including measures like enhanced hand hygiene and environmental cleaning, may be more effective at preventing VRE transmission.