[Objective] To explore and analyze the effect of disclosing agent methylene blue on bacterial culture results of different bacteria in vitro. [Methods] Eight isolates of reference strains of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Streptococcus pyogenes, and Candida albicans were incubated on the plates. Each bacterial suspension was formed by 50-fold dilution before the test methylene blue (MB) solution was added. For each strain, bacterial suspension was divided into 3 groups (5 samples each) exposed either MB solution 0.1%, MB solution 0.05% or sterile non-bacteriostatic 0.45% saline. The antimicrobial property of MB solution was determined by measuring the bacterial density on agar plates incubated for 24h and comparing it with controls exposed to sterile non-bacteriostatic 0.45% saline. [Results] Of the MB 0.01% or MB 0.05% exposure, reference strains but no Staphylococcus hominis and Acinetobacter baumannii strains resulted in fewer colony-forming units compared with the sterile saline control. The bacterial density of Acinetobacter baumannii on agar plates were significantly inhibited by MB solution (P<0.001), which were correlative with concentration. The colony-forming units of Staphylococcus hominis was also fewer (P<0.001), but there was no statistical difference between two concentrations of MB solution. [Conclusion] MB has a certain antibacterial effect on some pathogenic bacteria in periprosthetic joint infection.