Surgical site infection is a common complication after surgery. It is necessary to prevent or treat postoperative infection with antibiotics. The use of antibiotics could disturb the composition and/or functions of gut microbiota. The purpose of the present study was to explore the effect of antibiotics on gut microbiota in patients who underwent cardiac surgery. A total of 622 fecal samples were collected from 244 cardiac surgery patients. The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced on a MiSeq PE300. The gut microbiota diversity of samples was analyzed from alpha diversity at the OTU level and the distribution proportion of dominant bacteria at the genus level by a Circos graph, and the differences among different groups were compared by partial least squares discriminant analysis at the OTU level. As expected, antibiotics could perturb the composition of the gut microbiota in cardiac surgery patients. When antibiotics were administered over 7 days, the composition of the gut microbiota was significantly disturbed. The gut microbiota composition of patients returning to preantibiotic levels might need antibiotic withdrawal for at least 28 days.