Alpha-mangostin (α-MG) has been reported to be an effective antibacterial agent against planktonic cells of many Gram-positive bacteria. However, the antibiofilm potency of α-MG remains unexplored till date. In this study, the antibiofilm and mature biofilm eradication ability of α-MG against Staphylococcus epidermidis RP62A (ATCC 35984) biofilms were evaluated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of α-MG against S. epidermidis RP62A were found to be 1.25 and 5 μg/mL, respectively. α-MG exhibited a phenomenal concentration dependent rapid bactericidal activity (>4-log reduction within 5 min). In a multi-passage resistance analysis using S. epidermidis, no development of resistance to α-MG as well as antibiotics was observed in its habituation. α-MG at its 1/2 MIC effectively inhibited the initial biofilm formation of S. epidermidis, which was further confirmed through scanning electron microscopic (SEM) analysis that portrayed a lucid reduction in the aggregation and the spread of biofilm. The crystal violet staining and viable cell quantification results confirmed the eradication of preformed immature and mature biofilms of S. epidermidis by α-MG in a concentration dependent manner. Besides, the biofilm eradication ability was also confirmed through SEM and live/dead BacLight staining using confocal laser scanning microscopy (CLSM). Thus, the present study exemplifies that α-MG could plausibly assist to eliminate biofilm infections associated with multidrug-resistance staphylococci.