The influence of the anaerobic intestinal protozoan Blastocystis on human gut health remains poorly understood, particularly in type 2 diabetes mellitus (T2DM). A high prevalence of Blastocystis infection has been reported in T2DM patients, suggesting a potential link with gut microbiota. However, this association is not well documented in clinical settings. Hence, a comparative cross-sectional study was conducted involving 203 participants, including both T2DM patients and non-diabetic (NonDM) individuals, to evaluate the prevalence of Blastocystis subtypes associated with gut microbiota. Blastocystis subtypes were identified using polymerase chain reaction (PCR) and the faecal microbiome was analysed by targeting the V4 region of the bacterial 16S ribosomal gene. Our findings revealed that the prevalence of Blastocystis was higher in T2DM (25.49%) compared to NonDM (17.82%). The most common subtypes (STs) in the overall population were ST3, followed by ST1 and ST2. Notably, a significant change in the composition of gut microbiota was discovered between T2DM and NonDM with the NonDM group exhibiting higher alpha diversity based on species richness and diversity index. Furthermore, it was discovered that in both T2DM and NonDM, the presence of Blastocystis was significantly linked to increased alpha diversity. At the phylum level, the T2DM group showed a notable increase in Bacteroidetes and a marked decrease in Actinobacteria in the presence of Blastocystis compared with the negative Blastocystis group. Additionally, our genus profile analysis revealed that different Blastocystis subtypes exhibited a significant abundance of genera such as Bacteroides, Prevotella 9, Bifidobacterium, Blautia and Megamonas. Specifically, the abundance of Lentisphaerae and Tenericutes in less dominant phyla was significantly increased in Blastocystis ST1 and ST3 compared to other STs. Interestingly, a limited sample infected by Blastocystis ST7 was associated with an increase in pathogenic bacteria like Escherichia-Shigella in individuals with T2DM. These findings suggested that different Blastocystis subtypes may have contributed to a healthier gut microbiota or potentially led to dysbiosis. However, advanced analysis at the subtype (ST) level is necessary to elucidate the specific role of Blastocystis subtypes in gut health, as they may exhibit both commensal and pathogenic characteristics.