The susceptibility of Pyropia haitanensis and its bacteria to chloramphenicol, oleandomycin and cefotaxime was studied. They were combined with ampicillin, kanamycin and gentamicin to optimize the sterilization effect of P. haitanensis thalli. The effects were evaluated by plate count, qRT-PCR and 16S rRNA sequencing analysis. The results show that when P. haitanensis thalli was treated with ampicillin (300 mg/L), kanamycin (100 mg/L) and gentamicin (100 mg/L) mixture for 18 h, and then treated with chloramphenicol (50 mg/L), cefotaxime (200 mg/L) and oleandomycin (50 mg/L) mixture for 4 days, the health rate of the thalli is above 96.3%, and the inhibition rate of bacteria reached 99.9%. According to the results of qRT-PCR and 16S rRNA, the number of bacteria in this antibiotic combination was nearly reduced by half as compared with the first three antibiotic combination, and the bacteria abundance and diversity index were also significantly decreased. Among them, Pseudoalteromonas, Altererythrobacter, Salinimonas and Marinobacter were specifically inhibited. These results indicated that the optimal combination of multiple antibiotics could significantly affect the community structure of epiphytes on the thalli, which could be inhibited to the minimum, but still could not achieve the absolute sterilization.