Abstract: Grass carp (Ctenopharyngodon idella) were fed with basic feed (control group), basic feed supplemented with enrofloxacin or florfenicol for two weeks, respectively. Thereafter, enzyme activity, real time quantitative PCR (qRT-PCR), and high-throughput sequencing were used to analyze the effects of these two antimicrobials on the intestinal oxidative stress, immunity and intestinal microflora of C. idella. The results showed that, (1) additions of enrofloxacin and florfenicol could reduce the content of glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) in the intestines of C. idella, while they increased the amount of malondialdehyde (MDA), resulting in oxidative stress in the intestines of C. idella. (2) In the florfenicol group, the mRNA expressions of immune related factors, including TNF-α, IL-1β, IL-12, NF-κB-p65, sIgM and intestinal mucosa-related protein ZO-1, ZO-2, occludin, CLDN-1 and JAM3 were significantly lower than those in the control group. In the enrofloxacin group, the mRNA expressions of immune related factor, including TNF-α, IL-1 β, IL-12, TLR4, MHC Ⅱ and intestinal mucosa-related protein ZO-2, occludin, CLDN-1 and JAM3 were significantly lower than those in the control group. (3) The results of high throughput 16S rRNA sequencing showed that the additions of antimicrobials had no significant effects on α diversity (P > 0.05) of microflora in C. idella intestine. However, there were significant changes in β diversity (P < 0.05). The results showed that neither of the two antibiotics affected the community richness, but they changed the diversity among the sample communities. Taken together, dietary enrofloxacin and florfenicol could cause oxidative stress, inhibit the intestinal immunity as well as affect the intestinal microflora of C. idella. This study provides a reference for the researches related to the prevention and control of C. idella diseases and the green and healthy culture of C. idella.