Abstract: Two Thermophilic bacteria which exhibit antimicrobial activity were isolated from hot spring Jordan Valley. Those Gram-negative rods shaped were found to be Yersinia sp.1 and Aeromonas hydrophila. Extracts from aqueous phases of the tow strains were tested for antimicrobial activity by agar well diffusion method against 6 microorganisms (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterobacter sp. and Candida albicans). The highest antimicrobial activity was recorded by Yersinia sp.1 giving (16 mm) inhibition zone against Pseudomonas aeruginosa, where Aeromonas hydrophila produce no activity against the test organisms. Extracts from the two thermophilic strains showed relatively high antibacterial activity against Staphylococcus aureus. Partial purification of antimicrobial agents by Thin Layer Chromatography (TLC) gave two inhibitory bands with Rf values of 0.22 for extract from Yersinia sp.1 and 0.25 for extract from Aeromonas hydrophila.