Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disease that affects approximately 11% of the general population. The gut microbiota, among other known factors, plays a substantial role in its pathogenesis. The study aimed to characterize the gut microbiota differences between patients with IBS and unaffected individuals, taking into account the gender aspect of the patients and the types of IBS determined on the basis of the Rome IV Criteria, the IBS-C, IBS-D, IBS-M, and IBS-U. In total, 121 patients with IBS and 70 unaffected individuals participated in the study; the derived stool samples were subjected to 16S rRNA amplicon sequencing. The gut microbiota of patients with IBS was found to be more diverse in comparison to unaffected individuals, and the differences were observed primarily amongClostridiales,Mogibacteriaceae,Synergistaceae,Coriobacteriaceae,Blautiaspp., andShuttleworthiaspp., depending on the study subgroup and patient gender. There was higher differentiation of females’ gut microbiota compared to males, regardless of the disease status. No correlation between the composition of the gut microbiota and the type of IBS was found. Patients with IBS were characterized by more diverse gut microbiota compared to unaffected individuals. The gender criterion should be considered in the characterization of the gut microbiota. The type of IBS did not determine the identified differences in gut microbiota.