Background: Chemotherapy is the primary treatment modality used for patients with acute lymphoblastic leukemia (ALL), but inevitably causes microbiota-related oral complications. This study aims to investigate the effects of chemotherapy on oral health status, caries risk, and oral microbiome in pediatric patients with ALL. Methods: Thirty-nine children with ALL receiving chemotherapy were enrolled, and a gender-, age-, dentition stage and socioeconomic class matched healthy counterpart (HC) were recruited. Demographic information and overall health condition were obtained through questionnaire or medical records. Oral examination was performed to assess caries and salivary status, plaque index and other oral manifestations. Cariogram was used to assess the overall caries risk. Supragingival samples of thirteen ALL subjects and their counterparts were randomly selected to perform a 16S ribosomal RNA gene 454 pyrosequencing. Raw sequence data were screened, trimmed, and filtered using Seqcln and MOTHUR. Results: The prevalence of dental caries, gingivitis, oral mucositis, xerostomia and candidiasis in ALL group was higher than that of control group (p < 0.05). Children with ALL demonstrated higher caries risk compared with healthy controls based upon Cariogram (p < 0.05). The oral microbial structure of ALL patients receiving chemotherapy is different from that of healthy controls. Oral microbiota of ALL group showed less alpha diversity and significant differences in the composition of the oral microbiome compared to healthy controls. Conclusions: ALL patients receiving chemotherapy demonstrated compromised oral health, high caries risk, alteration of caries related factors and dysbiosis of oral microbiota. These findings may possibly be of clinical importance in developing better strategies for personalized preventive management of oral diseases for pediatric children with ALL.