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To explore the application of chitosan–gentamicin conjugate (CS-GT) in inhibiting <i>Vibrio parahaemolyticus</i> (<i>V. parahaemolyticus</i>), which is an important pathogen in aquatic animals worldwide, the antimicrobial activity of CS-GT and the effects of a CS-GT dose on the intestine histopathology and intestinal flora of <i>V. parahaemolyticus</i>-infected shrimps were explored. The results showed that CS-GT possessed broad-spectrum antibacterial activity, with minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and half inhibitory concentration (IC₅₀) of 20.00 ± 0.01, 75.00 ± 0.02 and 18.72 ± 3.17 μg/mL for <i>V. parahaemolyticus</i>, respectively. Further scanning electron microscope and cell membrane damage analyses displayed that the electrostatic interaction of CS-GT with cell membrane strengthened after CS grafted GT, resulting in leakage of nucleic acid and electrolytes of <i>V. parahaemolyticus</i>. On the other hand, histopathology investigation indicated that high (100 mg/kg) and medium (50 mg/kg) doses of CS-GT could alleviate the injury of a shrimp’s intestine caused by <i>V. parahaemolyticus</i>. Further 16S rRNA gene sequencing analysis found high and medium dose of CS-GT could effectively inhabit <i>V. parahaemolyticus</i> invasion and reduce intestinal dysfunction. In conclusion, CS-GT possesses good antibacterial activity and could protect shrimps from pathogenic bacteria infection.