Staphylococcus aureus is widely distributed in environment and can cause various human infection and food poisoning cases. Also, this pathogen is a typical biofilm former, which further complicates its pathogenicity. Antibiotics have been widely used to eliminate pathogenic bacteria, but their indiscriminate use has also led to the widespread emergence of drug-resistant bacteria, such as Methicillin-Resistant Staphylococcus aureus (MRSA). In this study, the effect of antibiotics on biofilm formation of MRSA strains 875 and 184 was explored. Firstly, MRSA 875 belongs to SCCmec type IV, ST239, carrying the atl, icaA, icaD, icaBC, and aap genes, and MRSA 184 belongs to SCCmec type II, ST5, carrying the atl, icaD, icaBC, aap, and agr genes. Then, a total of 8 antibiotics have been selected, including kanamycin, gentamycin, cipprofloxacin, erythromycin, meropenem, penicillin G, tetracycline, vancomycin. Minimum inhibitory concentrations (MICs) of each antibiotic were determined, and MIC of MRSA 875 and 184 to kanamycin/gentamicin are 2048/64 μg/mL and 2048/4 μg/mL, respectively. A total of 10 concentrations, ranging from 1/128 to 4 MIC with 2-fold, were used to study biofilm formation. Biofilm biomass and viability were determined during different phases, including initial adhesion (8 h), proliferation (16 h), accumulation (24 h) and maturation (48 h). Importantly, kanamycin at specific concentrations showed significant promotion of biofilm biomass and biofilm viability, with none of such observation acquired from other antibiotics. This study provides scientific basis and new research ideas for the quality control technology of microorganisms and safety prevention of MRSA.