Two extract from the walnut tree bark (Juglans regia) were investigated for antimicrobial activities against seven human pathogens such as: Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, salmonella suis ATCC 13076, Pseudomonas aeruginosa ATCC 27583, Escherichia coli ATCC 25922 and Shigella sonnei ATCC 11060 and one was human pathogenic fungus: Candida albicans ATCC 10231. In this study, methanol was used as an extraction solvent, and bioactivity screening was done by agar well diffusion method. The bark extract in both samples showed strong activities against almost all the pathogenic microorganisms. Furthermore, the purified crude extract of the two samples has been analyzed using gas chromatography-mass spectrometry (GC-MS). Results indicated that the main component in the crude extract for bark sample 1 (MHO) was cyclobutanol (91.7%) and for bark sample 2 (MHC) was 1, 3-dioxolane, 2, 4, 5-trimethyl (92.9%). In both extracts cyclobutanol was present in high percentage (91.7%). Further, 4h-pyran-4-one,2,3-dihydro-3,-dihydroxy-6-methyl; 1,3-dioxolane-4-methanol,2,2-dimethyl; 1,6,3,4-dianhydro-2-deoxy-beta-d-lyxo-hexopyranose; cyclopropyl carbinol; 2-heptanamine,5-methyl-; pyrimidine-2,4(1h,3h)-dione,5-amino-6-nitroso; alanine; butanenitrile,2,3-dioxo-,dioxime,o,o-diacetyl; (r)-(-)-2-amino-1-propanol and benzaldehyde,4-ethoxy were present in both bark extracts as 80-89%. Moreover, ethanamine,2-propoxy; 2-isopropoxyethylamine; (r)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol; 3,4-furandiol, tetrahydro-,cis; glycerin; eicosanoic acid; pentadecanoic acid; 1-octadecanamin,n-methyl and (s)-(+)-1-cyclohexylethylamine appeared in bark sample 1 as 80-91%, while the compounds 1,3-dioxolane,2,4,5-trimethyl; 11-tridecen-1-ol; hexadecanal; 4-tridecene,(z); 2-undecene,(e); 4-tetradecene,(z); acetic acid, trifluoro-, decyl ester; 2-hydroxy-3,5-dimethylcyclopent-2-en-1-one and d-glycero-d-tallo-heptose were present in bark sample 2 as 80-92%.