During its traditional preparation, the formula is prepared by extraction with freshly prepared coconut milk and low-temperature cooking to obtain an active oil. Although our previous work indicated an anti-biofilm activity of the ethanol extract of YaSP, the anti-biofilm capability of a traditionally prepared form of YaSP has not been proven yet. Therefore, this study aimed to investigate the anti-biofilm activity of YaSP prepared from both oven-dried and fresh plant parts. Garcinia mangostana and α-mangostin were additionally tested as active ingredients of YaSP. This study revealed that a poly-herbal formula, YaSP, remarkably disturbs P. aeruginosa biofilm formation and affects its biofilm architecture. The eradication of P. aeruginosa mature biofilms after treating with YaSP was also demonstrated.