In the cultivation of freshwater nucleated pearls, the treatment technology of nucleated pearls is very important for the quality and yield of pearls. Pearl nuclei treated with astragalus extract, flavomycin, and oxytetracycline were found to possess anti-inflammatory and antibacterial effects depending on drug concentrations. To optimize the concentration of various drugs for the preparation of medicinal pearl nuclei to enhance pearl production Staphylococcus aureus and Aeromonas hydrophila were used as experimental strains. A comprehensive antibacterial circle diameter was taken as an indicator of drug efficacy. The response surface method based on a central composite design (CCD) was used to explain the combined effect of drugs and optimize their concentrations. The optimal concentrations of flavomycin, oxytetracycline, and astragalus extract were 9.10, 10.50, and 1.50 g/mL, respectively. In these conditions, the predicted antibacterial circle diameter was 1.83 cm and the measured value was 1.81 cm, accounting for a 0.5% relative error. The pearl nuclei coated with the abovementioned drug combinations had a strong antibacterial effect on both A. hydrophila and S. aureus, thus establishing a basis for the development and application of medicinal pearl nuclei.