Data concerning the human microbiota composition during Clostridioides (Clostridium) difficile infection (CDI) using next-generation sequencing are still limited. We aimed to confirm key features indicating tcdB positive patients and compare the microbiota composition between subgroups based on toxin gene load (tcdB gene) and presence of significant diarrhea. Ninety-nine fecal samples from 79 tcdB positive patients and 20 controls were analyzed using 16S rRNA gene sequencing. Chao1 index for alpha diversity were calculated and principal coordinate analysis was performed for beta diversity using Quantitative Insights into Microbial Ecology (QIIME) pipeline. The mean relative abundance in each group was compared at phylum, family, and genus levels. There were significant alterations in alpha and beta diversity in tcdB positive patients (both colonizer and CDI) compared with those in the control. The mean Chao1 index of tcdB positive patients was significantly lower than the control group (P<0.001), whereas there was no significant difference between tcdB groups and between colonizer and CDI. There were significant differences in microbiota compositions between tcdB positive patients and the control at phylum, family, and genus levels. Several genera such as Phascolarctobacterium, Lachnospira, Butyricimonas, Catenibacterium, Paraprevotella, Odoribacter, and Anaerostipes were not detected in most CDI cases. We identified several changes in the microbiota of CDI that could be further evaluated as predictive markers. Microbiota differences between clinical subgroups of CDI need to be further studied in larger controlled studies.