The rise in resistant infections, which are primarily brought on using antibiotics carelessly, is a serious issue for the community. This study aimed to examine the antibiofilm and antibacterial activity of Populus euphratica ethanolic extract against the resistance of Methicillin-resistant Staphylococcus aureus (MRSA) isolates. Leaves of Populus euphratica were collected and extracted using the Soxhlet apparatus. Active chemical compounds were detected by some reagents and screened using Gas chromatographic–Mass spectral (GC-Mass) analysis. Fifteen clinical MRSA isolates from several different clinical samples were detected for biofilm formation, and an antibiotic susceptibility test was performed to investigate the sensitivity pattern. Minimal Inhibitory Concentration (MIC and Sub MIC) for most sensitive antibiotic and Populus euphratica extract were investigated. The results showed that this plant has a greater percentage of glycosides and polyphenolic compounds than other compounds. In GC/MS analysis, twenty-six compounds have been detected; 8-Octadecenoic acid methyl ester(E) was the most abundant compound (13.98 %), followed by Eicosane (10.77%). On the other hand, the ethanolic extract of  Populus euphratica has a good antibacterial effect against MRSA isolates in a concentration-dependent manner. The study also revealed that Populus euphratica was highly effective against biofilms in comparison with the most sensitive antibiotic (Ciprofloxacin). In conclusion, the studied plant can be used as an alternative and natural defense material against chronic infections caused by bacterial biofilms.