Still there is percentage of HCV patients not responding to Direct Acting Antiviral Agents (DAAS), even the responder HCV patients are in need to follow up. Gut flora (Microbiota) include all the microorganisms in the intestine and liver can be greatly affected by changes in gut microbiota. The study was done to evaluate the association between gut flora and the response to DAAS in chronic HCV patients. Two groups; group 1 (No=15 of HCV responders patients) and group 2 (No=15 non responder HCV patients) treated by DAAS according to the treatment protocol of the Egyptian National Committee for Control of Viral Hepatitis (NCCVH). Healthy control subjects (No=15) age and sex matched to the study groups as third group. Full history taking, clinical examination and all investigations were done plus stool culture and 16srRNA gene amplification and sequencing were done.The results show statistically significant difference between the patients (responders and non-responders) and control subjects .Microbiota which decreased in non-responder group than responder group (Lactobacillus brevis strain with P value: 0.013*, Pediococcus pentosaceus with P value: 0.014*, Clostridium tetani with P value: 0.006*, Shigella flexneri with P value: < 0.001*, Shigella dysenteriae P value: 0.008*, Shigella sonnei with P value: 0.026*). Microbiota which increased in non-responder group than responder group (Pseudomonas aeruginosa with P value: 0.003*, Streptococci with P value: 0.002*). Microbiota which increased in responder group than non-responder and control groups (Enterobacter hormaechei with P value: 0.013* and Enterococcus fecalis with P value: 0.01*). In conclusion, the analysis of stool samples by using 16S rRNA gene of patients with chronic HCV infection (responders to DAAs and non-responders) in comparison to healthy individuals is important issue. Patients with HCV had a few significant changes that may be related to liver-controlled homeostasis, protein synthesis, lipid digestion, or possibly to bacterial translocation, immune modulation, or a combination of all of the above mechanisms than healthy individuals. Even the responder patient needed to be followed up to modulates his microbiota changes to his health state. Non-responder group needs strict observation and modulation for their microbiota to be similar to the responder group and to avoid the development of HCC.