The increasing prevalence of multidrug-resistant (MDR) pathogens necessitates the discovery of novel antibacterial agents. In this study, culturable soil bacteria from the Dead Sea region (Jordan) were isolated and the antibacterial activity of their extracellular metabolites were evaluated. Twenty-one isolates were screened using a perpendicular-streak primary assay; three stable producers (GH-15, GH-20, GH-21) were prioritized. Cell-free supernatants were extracted with n-hexane and ethyl acetate, tested using agar well diffusion, and profiled using thin-layer chromatography (TLC) coupled with bioautography (GH-15). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution against Bacillus subtilis ATCC 11774, B. cereus ATCC 10876, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Proteus mirabilis ATCC 29906. The ethyl acetate extract of GH-15 showed the lowest MIC against E. coli (0.073 mg/mL). The MIC values were equal to MBC for several strains, indicating bactericidal activity. For GH-15 hexane extract, the lowest MIC was 2.625 mg/mL (E. coli and B. cereus), and the lowest MBC was 5.25 mg/mL (E. coli and B. cereus). Dead Sea soils yielded bacterial isolates with broad antibacterial activity; solvent-dependent activity and TLC-bioautography suggested multiple bioactive metabolites, supporting further purification and chemical identification.
