Background There is growing evidence that eosinophilic infiltration can release mediators which are harmful to the intestinal epithelium in patients with irritable bowel syndrome (IBS). Although cyclooxygenase 2 (COX-2) and nuclear factor-kappa beta (NF-kB) expression had been previously reported to increase in many inflammatory conditions, there is a paucity in data investigating their expressions in IBS. Our aim was to evaluate colonic mucosal eosinophilia and immunohistochemical expression of COX-2 and NF-kB in patients with irritable bowel syndrome. Methods A total of 80 patients who met the inclusion criteria of IBS based on Rome IV symptoms questionnaire were subjected to abdominal ultrasound, laboratory investigations, serum immunoglobulin E (IgE) level assessment and colonoscopic examination. Immunohistochemistry was performed to detect COX-2 and NF-kB expression in colonic biopsies obtained from IBS patients. Results Histopathological examination showed that 60 colonic biopsy specimens (75%) showed few mixed inflammatory cells ≤3 cells/ HPF, 12 biopsy specimens (15%) showed eosinophilic infiltration ≥25 eosinophils/HPF and 8 biopsy specimens (10%) showed severe lymphocytic infiltration and aggregation. Colonic eosinophilic infiltrate was significantly higher among patients presented with IBS-D subtype. Serum IgE was significantly higher among patients with colonic eosinophilic infiltrate than the others. In IBS-D patients, colonic mucosa showed positive expression of COX-2 and NF-kB in 52.1% and 81.25% of cases, respectively. Conclusion Patients with IBS -particularly IBS-D subtype- should undergo colonoscopy and biopsy to exclude underlying inflammatory pathology. Moreover, patients with positive COX-2 and NF-kB need further evaluation and follow-up.