Antibacterial effect of tilmicosin on 3 kinds of aquatic pathogenic bacteria, control effect of tilmicosin against the infection by Aeromonas hydrophilia to Carassius auratus gibelio, and toxic effect of tilmicosin on Carassius auratus gibelio were investigated. Eleven mass concentrations (0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 mg/L) of tilmicosin were designed, and the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) of tilmicosin against Aeromonas hydrophilia, Aeromonas sobria and Vibrio anguillarum were observed, and the ratios of MBC and MIC were calculated. Carassius auratus gibelio with the weight of（217.65±25.26）g were infected by intraperitoneal injection of 0.3 mL of Aeromonas hydrophilia at a concentration of 9×108 CFU/mL, then they were given by oral administration of tilmicosin with the dosages of 0, 50, 100 and 150 mg/kg for three consecutive times at 1, 24 and 48 h, respectively, and protection rate of tilmicosin for Carassius auratus gibelio injected by Aeromonas hydrophilia was calculated. Six dosages of tilmicosin (0, 260, 300, 340, 387 and 440 mg/kg) were given to Carassius auratus by oral administration, respectively. Mortality rate was recorded, and the median lethal dose (LD50) was calculated. The MIC for Aeromonas hydrophilia, Aeromonas sobria and Vibrio anguillarum were 4.00, 0.50 and 1.00 mg/L, respectively, the MBC were 64, 32 and 32 mg/L, and the ratios of MIC and MBC for Aeromonas hydrophilia, Aeromonas sobria and Vibrio anguillarum were 16, 64 and 32, respectively. The doses of 100 and 150 mg/kg of tilmicosin were both effective for protecting the Carassius auratus gibelio infected by Aeromonas hydrophilia. The LD50 at 24 h, 48 h, 72 h and 96 h were 344.0, 306.3, 298.5 and 298.5 mg/kg, respectively. Tilmicosin showed the antibacterial effect in vitro to some extent, and the protective effect of Carassius auratus gibelio infected by Aeromonas hydrophilia. But it also showed toxic effect for Carassius auratus gibelio. It is helpful for the further development and utilization of tilmicosin in the field of aquaculture.