There is evidence linking hand, foot and mouth disease (HFMD) to gut microbiota dysbiosis, and this relationship was corroborated in a large HFMD patient population in our previous study. Here, we present a bacterial 16S rRNA gene dataset from faecal samples of 713 individuals (254 HFMD patients, 459 healthy controls) aged 2 to 7 years residing in Heyuan and Jiangmen counties, Guangdong Province, southern China. Microbiome analysis indicated a significant increase in genus Prevotella, Cetobacterium, and Megamonas was observed in patients with HFMD, whereas a large increase in genus Bacteroides, Ruminococcus, and Faecalibacterium were seen in the control group. We also share the bioinformatic analytical pipeline for this analysis, from data preprocessing to data filtering and amplicon sequence variant (ASV) table generation. We expect that the dataset will be reprocessed, evaluated and fully analysed with various analysis methods to further elucidate the role of the gut microbiota in HFMD development.