OBJECTIVE: Given the unclear relationship between Blastocystis spp. infection and colorectal cancer (CRC), this study aimed to provide insight into Blastocystis infection, assess the effect of chemotherapy on Blastocystosis in CRC patients, and explore potential links between CRC and Blastocystis subtypes (STs). SUBJECTS: A total of 150 participants were divided into three groups: Group I (50 CRC patients not receiving chemotherapy), Group II (50 CRC patients who were receiving chemotherapy), and Group III (50 healthy, age- and sex-matched controls). RESULTS: Blastocystis spp. was detected in 42 cases through microscopy and culture, with infection rates higher in Group I (40%) and Group II (32%) compared to the control group (12%). Among all participants, 86 were asymptomatic, while 64 experienced symptoms. PCR analysis confirmed Blastocystis in 26 out of the 42 cases. PCR-restriction fragment length polymorphism (RFLP) analysis identified 60% of isolates as Group A and 16% as Group C, while sequencing later confirmed that 24% belonged to Group B. PCR-sequence-tagged site (STS) analysis revealed five STs (ST1, ST2, ST3, ST5, and ST7), with ST1 (52%) and ST3 (24%) identified as the most prevalent STs. CONCLUSION: Blastocystis infection was significantly higher in CRC patients, suggesting a possible association with the disease. It appears to act as an opportunistic pathogen, contributing to symptom development regardless of CRC. The absence of significant differences in ST distribution across groups indicates that Blastocystis pathogenicity is complex and not exclusively linked to specific STs.