Previous studies have shown that bioactive glasses possessed antibacterial effect on common bacteria due to the high aqueous pH value caused by the bioactive glass dissolution. In the present study, the efficiency of the antibacterial effect of 45S5 Bioglass (45S5 BAG) against S. aureus, S. epidermidis and E. coli and its mechanism were investigated. The results showed that 45S5 BAG exhibited a strong antibacterial effect against the bacteria, and the sensitivity of gram-negative and gram-positive bacteria to Bioglass was different. Furthermore, a dose-dependent bacterial adhesion on 45S5 BAG particles and the formation of needle-like Bioglass debris were observed, which resulted in the damage of cell walls and inactivation of bacteria. The results suggested that both the high pH and bioglass debris on the surface of bacteria may be the possible mechanisms of the antibacterial effect of 45S5 BAG particulates.