OBJECTIVE: To define the temporal relationship between intrapartum intravenous vancomycin administration and vaginal group B streptococcus (GBS) colony counts. STUDY DESIGN: Prospective cohort study conducted from October 2014 to February 2017. Women with antenatal cultures demonstrating GBS colonization and a plan for vancomycin administration were eligible. Intrapartum vaginal cultures were collected prior to the first vancomycin infusion and every 2 hours up to five collections or delivery. Results were analyzed in two groups: participants with at least one positive intrapartum culture and those without any positive intrapartum cultures. RESULTS: A total of 63 women were enrolled. Among consented women, a total of 8 were excluded and 3 participants' cultures were never plated, thus leaving a total of 52 women for analysis. The degree of vaginal GBS colonization varied between subjects and was not normally distributed. Colony counts dropped rapidly from hour 0 to hour 2 (median: 6.0 × 108 vs. 1.0 × 108, p < 0.01). Standardizing hour 0 colony counts to 100%, the percent decline in colony counts from hour 0 to hour 2 was significant (p = 0.03), and at each subsequent time point fell further. CONCLUSION: GBS vaginal colony counts fall rapidly after intrapartum vancomycin administration.