An emerging evidence suggests that microRNAs (miRNAs) may be reliable biomarkers for inflammation or oncogenesis. The aim of this study was to investigate the diagnostic value of miR-23a and miR-181b in patients with irritable bowel syndrome (IBS) and patients with colorectal cancer (CRC) vs. healthy controls. Forty patients with IBS (29 females, 11 males), 33 patients with CRC (14 females, 19 males), and 33 healthy controls (17 females, 16 males) were prospectively included. Serum levels of miRNAs were evaluated by quantitative reverse transcription PCR. MiR-23a and miR-181b had significantly higher serum levels (p = 0.0009 and p = 0.004, respectively) in IBS patients than in controls. Serum levels of miR-23a and miR-181b in CRC patients were also significantly higher than in controls (p = 0.002 and p = 0.029, respectively). The levels of miR-23a and miR-181b in CRC vs. IBS patients suggested a trend of overexpression in patients with CRC, however, without statistical significance (p = 0.169 and p = 0.179, respectively). miRNet and Reactome database showed phosphatase and tensin homolog (PTEN) to be a major common pathway, suggesting inflammation as a central mechanism. MiRNAs may serve as reliable biomarkers in clinical practice, but further studies are necessary to establish a cut-off limit for specific miRNAs in different diseases.