Oleoresin of Abies balsamea (L.) Mill. was used by Native Americans of the boreal forest of Canada and French Canadians to treat various infections, suggesting that oleoresin has antibacterial properties. In this study, the antibacterial activity of whole oleoresin from A. balsamea was investigated against E. coli, S. aureus and two methicillin-resistant S. aureus (MRSA) strains using a new sensitive assay developed to evaluate hydrophobic matrix and compounds. Antibacterial activity of oleoresin was first investigated using dilution and disk diffusion methods against E. coli and S. aureus, and compared to a new sensitive assay for hydrophobic matrix. Moreover, whole oleoresin was analyzed by GC-MS to characterize the composition and to identify the compounds responsible of the antibacterial activity. The results showed that whole oleoresin was inactive against Gram-negative E. coli (MIC This study supports the use of oleoresin of A. balsamea by the Native Americans and French Canadians to treat bacterial infections due to S. aureus.