This study aimed to investigate the antibiofilm activity of alpha-mangostin (AMG) loaded nanoparticles (nanoAMG) against Staphylococcus aureus, including the methicillin-resistant strain MRSA252. The results indicated that treatment with 24 lmol/L nanoAMG inhibited the formation of biofilm biomass by 53– 62%, compared to 40–44% for free AMG (p < 0.05). At 48 lmol/L, biofilms in all nanoAMG treated samples were nearly fully disrupted for the two tested strains, MRSA252 and the methicillin-sensitive strain NCTC6571. That concentration resulted in killing of biofilm cells. A lower concentration of 12 mmol/L nanoAMG inhibited initial adherence of the two bacterial strains by > 50%. In contrast, activity of nanoAMG was limited on preformed mature biofilms, which at a concentration of 48 mmol/L were reduced only by 27% and 22% for NCTC6571 and MRSA252, respectively. The effects of AMG or nanoAMG on the expression of biofilm-related genes showed some noticeable differences between the two strains. For instance, the expression level of ebpS was downregulated in MRSA252 and upregulated in NCTC6571 when those strains were treated with either AMG or nanoAMG. In contrast, the expression of fnbB was down regulated in NCTC6571, while it was up-regulated in the MRSA252. The expression of other biofilm-related genes (icaC, clfB and fnbA) was down regulated in both strains. In conclusion, our results suggest that AMG coated nanoparticles had enhanced biological activity as compared to free AMG, indicating that nanoAMG could be a new and promising inhibitor of biofilm formation to tackle S. aureus, including strains that are resistant to multiple antibiotics.