BACKGROUND: Hand, foot, and mouth disease (HFMD) is a prevalent infectious condition in children. This study aimed to assess the regulatory effects of Re-Du-Ning on the intestinal microflora of pediatric patients with HFMD. METHODS: Fecal samples were collected from children affected by HFMD, who were diagnosed at the traditional Chinese medicine pediatrics outpatient and emergency departments of Liuzhou Women and Children's Healthcare Hospital, as well as from healthy children undergoing physical examinations at the same hospital during the same period. DNA was extracted from these samples and subjected to 16S ribosome DNA amplicon sequencing. The sequenced data were categorized, quantified, and compared. Analyses involved creating relative abundance bar graphs, constructing unweighted pair-group method with arithmetic mean clustering trees, and generating heatmaps of clustering to evaluate the variations in abundance and diversity across different groups. The analysis of molecular variance and -test were used to analyze structural differences in microbial flora between groups, and linear discriminant analysis was used to identify significant differences in microbial genera between the groups. RESULTS: A total of 67 fecal samples were collected from children with HFMD (13 in the intravenous group, 40 in the enema group) and from healthy children (14 in the healthy group). When compared with the healthy group, the intestinal microflora diversity and similarity were highest after enema treatment, although the microbial structure exhibited significant changes (weighted_unifrac, P<0.05). The composition of species relative abundance was comparable between the healthy group and the post-enema group. CONCLUSIONS: Re-Du-Ning enema treatment regulated the intestinal microflora in these children, significantly increasing the abundance of probiotics like and reducing the abundance of opportunistic pathogens like .