As a cause of vulvovaginitis in prepubertal girls, Streptococcus pyogenes (S. pyogenes) is commonly isolated from vaginal introitus swabs. Studies have identified several risk factors, but have not focused on the correlation between ambient air pollutants and S. pyogenes-induced vulvovaginitis in prepubertal girls. This study was conducted to determine whether ambient air pollutants were associated with S. pyogenes-induced vulvovaginitis in prepubertal girls. Daily data about S. pyogenes-induced vulvovaginitis in prepubertal girls from the outpatient department of Children’s Hospital at the Zhejiang University School of Medicine in Hangzhou City between January 1, 2015, and December 31, 2018, were retrospectively reviewed. Ambient air pollutants in Hangzhou were measured daily. A generalized additive model (GAM) was utilized to assess the associations between daily air pollutants and S. pyogenes isolates obtained from vaginal introitus swabs of prepubertal girls. The mean daily concentration of nitrogen dioxide (NO₂) in Hangzhou City during the study period was 44.6 μg/m³ (25th–75th percentiles, 32.0–56.0 μg/m³). The GAM showed that the largest estimate effects in S. pyogenes-induced vulvovaginitis in prepubertal girls were found in NO₂ with a moving (accumulative) average on day 3. The excess risk of NO₂ in terms of the daily number of S. pyogenes isolates obtained from the vaginal introitus swabs was 14.91% (95% confidence interval [CI]: 4.85–25.94%) in the single-pollutant model. The multipollutant model revealed that an increase of 10 μg/m³ in NO₂ exposure was associated with an 18.33% increased risk for acquiring S. pyogenes-induced vulvovaginitis in prepubertal girls (95% CI: 1.21–38.35%; P < 0.05). In conclusion, short-term NO₂ exposure was strongly associated with the spread of S. pyogenes-induced vulvovaginitis in prepubertal girls.