To study the bacteria and fungi causing vulvovaginitis in prepubertal girls. Swabs from vaginal introitus were collected from patients with vulvovaginitis in 2018, and cultured for the identification of microorganisms with standard microbiological techniques. A children's hospital in Hangzhou, East China. A total of 1235 Chinese prepubertal girls diagnosed with vulvovaginitis. Bacteria or fungi in pure cultures or as the predominant organism were defined as pathogens. A total of 1235 cases were diagnosed as vulvovaginitis, and 515 isolates were identified from 494 patients (40%, 494/1235). The most common pathogen was Streptococcus pyogenes (27.6%,142/515), followed by Haemophilus influenzae (27.2%,140/515), Candida albicans (22.3%,115/515), Staphylococcus aureus (5.8%, 30/515) and Escherichia coli (4.7%,24/515). All S pyogenes isolates were sensitive to penicillin, whereas 53.7% (73/136) of H influenzae isolates were sensitive to ampicillin, and 70.4% (19/27) of S aureus isolates were sensitive to oxacillin. S pyogenes and H influenzae were the 2 most commonly identified pathogenic bacteria found in prepubertal girls with vulvovaginitis. Vulvovaginitis in prepubertal girls caused by C albicans may be more common in older children.