Methanol extract obtained from the fruits of Hyphaene thebaica (doum fruit) was chemically analyzed using GC-MS (gas chromatography–mass spectrometry). Up to thirty compounds were identified in the extract. Acetic acid decyl ester (36.80%), n-Hexadecenoic acid (5.14%),1H-Purine-2,6-dione, 3,7-dihydro-1-methyl (4.24%), 2-Furancarboxaldehyde, 5-(2-hydroxy-2-phenylacetyl)-dimethylhydrazone (4.67%), Propanoic acid 3,3′-dithiobis (3.52%) and [1,2,4] Triazolo[1,5-a]pyrimidin-7-ol were major components. The antibacterial potential of the extract against six clinical bacterial isolates resistant to antibiotics was also investigated, using various in vitro assays including well diffusion, minimal inhibitory and minimal bactericidal concentration. It was found that, the methanol extract of doum fruit was characterized by antibacterial action toward one Gram-positive ß-lactamase bacteria (Staphylococcus aureus), and one Gram-negative Multidrug-resistant bacteria (Proteus mirabilis). The other four bacterial strains showed no susceptibility towards the extract. The study suggests future additional biochemical and microbiological investigations in order to understand the mechanism of action of the bioactive molecules as antimicrobial agents.