Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disease presenting clinically by abdominal pain with alteration of bowel habits. Although IBS has uncertain etiology, chronic gut inflammation due to persistent exposure to an infectious agent including Blastocystis sp. was proposed. The aim of this study was to detect the prevalence of Blastocystis sp. subtype (ST) isolated from stool of IBS patients and to assess Blastocystis sp. and H. pylori co-infection in IBS patients from Beni-Suef Governorate, Egypt. Stool samples were collected from 115 IBS patients, following Rome III criteria. All stool samples were microscopically examined by wet mount and permanent trichrome stain, cultured on Jones' medium with further sequencing of positive Blastocystis isolates and screened for detection of H. pylori coproantigen. Blastocystis sp. was the predominant parasite in IBS patients; it had statistical significant association with both rural residence (OR = 10) and flatulence (OR = 8.2). There was a predominance of Blastocystis sp. ST3 (81%) followed by ST1 (19%). Blastocystis culture results (19.1%) were superior than microscopy (16.5%). The majority of Blastocystis-positive IBS patients (72.7%) were co-infected with H. pylori with statistical significance; however, H. pylori was higher in Blastocystis-negative IBS patients (47/64) than in Blastocystis-positive IBS patients (17/64). Interestingly, IBS is usually associated with gut dysbiosis, while the most prevalent parasite in our IBS patients was Blastocystis sp., which is frequently found in asymptomatic individuals. Whether Blastocystis sp. is a cause or a consequence of IBS still needs further investigation, with a particular focus on correlation of IBS with different Blastocystis sp. subtypes and gut microbiomes.