Intestinal dysbiosis is key in the onset and development of Crohn’s disease (CD). We evaluated the microbiota changes in CD patients before and after a six-month anti-TNF treatment, comparing these changes with the microbiota of healthy subjects. This prospective multicenter observational study involved 27 CD patients initiating anti-TNF treatment and 16 healthy individuals. Inflammatory activity was determined at baseline, 3 and 6 months, classifying patients into responders and nonresponders. Fecal microbiota was analyzed by massive genomic sequencing thought 16S rRNA amplicon sequencing before and after six months of anti-TNF treatment. The CD cohort showed a decrease in genera of the class Clostridia, short-chain fatty acid producers, and an increase in the phylum Proteobacteria (p < 0.01) versus the healthy cohort. After anti-TNF treatment, the phylum Proteobacteria also increased in non-responders versus responders (13/27) (p < 0.005), with the class Clostridia increasing. In addition, alpha diversity increased in responders versus non-responders (p < 0.01), tending towards eubiosis. An association was found (p < 0.001) in the F.prausnitzii/E.coli ratio between responders and non-responders. The F/E ratio was the most accurate biomarker of anti-TNF response (area under the curve 0.87). Thus, anti-TNF treatment allows partial restoration of intestinal microbiota in responders and the F.prausnitzii/E.coli ratio can provide a reliable indicator of response to anti-TNF in CD.