Abstract: This study investigated the effects of penicillin and florfenicol on the histological structure, antioxidant activity, and intestinal microbiota of yellow drum. Different concentration groups were set for penicillin (25, 50, and 75 mg/kg) and florfenicol (25, 50, and 100 mg/kg), with a basic feed group as the control. Samples were collected on the 5th and 10th days to assess changes in intestinal and liver histology, antioxidant enzyme activity, immune gene expression, antibiotic residues in muscle, and alterations in intestinal microbiota composition. The results showed that: (1) Low and medium concentrations of penicillin or florfenicol did not significantly affect the intestinal and liver histology, but high concentrations caused clear damage to both tissues. (2) High concentrations of both antibiotics, regardless of treatment duration, significantly reduced liver SOD, CAT, and GSH-PX activities, while increasing MDA content. In the intestine, SOD activity decreased, while GSH-PX activity and MDA content increased. (3) Antibiotic residues were detected in both liver and muscle tissues. After 10 days of high-concentration treatment, the residual levels of penicillin (54.48 μg/kg) and florfenicol (1039.47 μg/kg) in muscle tissue exceeded the maximum residue limits. (4) High concentrations of penicillin or florfenicol significantly inhibited the expression of Arp2/3, ZO-1, and Claudin4 in the intestine, while pro-inflammatory IL-6 and TNF-α increased, and anti-inflammatory IL-10 and TGF-β decreased. 5) Neither penicillin nor florfenicol significantly affected the alpha diversity of intestinal microbiota, but Proteobacteria increased significantly, while Firmicutes decreased. In conclusion, low concentrations of penicillin or florfenicol had no significant effects on liver and intestinal structure, antioxidant enzyme activity, or expression of intestinal structural and immune genes, or microbiota composition. However, high concentrations caused liver and intestinal damage, oxidative stress, mucosal immune responses, and altered microbiota diversity and composition.