The emergence of antibiotic resistance has become a major concern, surpassing the development of new antibiotics. This has led to a post-antibiotic era where even minor injuries and common infections can become life-threatening. The misuse and overuse of antibiotics have further contributed to the rise of antimicrobial resistance (AMR). To tackle this challenge, it is crucial to explore bioactive metabolites from microbial sources and discover new antibiotic compounds for improved therapeutics against infectious diseases. The objective of the research mentioned was to identify antibiotics produced by a producer strain (DGSI), which was previously isolated from the Dead Sea region, in the search for a novel antibiotic. The identification of DGSI using the API kit has yielded the identification of Bacillus subtilis with a confidence level of 92.6%. However, through 16S rRNA sequencing, it was identified as Bacillus vallismortis (B. vallismortis) with a 99.63% match. This new strain was submitted to the NCBI GenBank as B. vallismortis DGSI strain under accession number OQ568809. The DGSI supernatant was extracted using n-hexane, and the resulting crude extract underwent purification through thin-layer chromatography (TLC) using a solvent system of chloroform and methanol in a ratio of 9.2:0.8. Using the bioautography method, the antimicrobial activity of the partially purified compound was determined, resulting in the discovery of two active compounds: compound 1 (Rf = 0.22) and compound 3 (Rf = 0.55). The study successfully isolated two distinct active compounds that exhibited potent antimicrobial properties. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for each compound. The results indicated that compound 3 demonstrated antimicrobial activity against both Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). On the other hand, the activity of compound 1 was limited to Gram-positive bacteria only. The structures of the two antibiotic compounds were identified based on their spectral data, including 1D, 2D nuclear magnetic resonance (NMR), and Fourier-transform infrared (FTIR) spectroscopy. The structural chemical analysis of compound 1 suggested that it is likely a lipopeptide or flavonoid, while compound 3 is most likely a peptide, although the possibility of it being a carbohydrate cannot be ruled out. However, determining the exact structures of the compounds proved to be challenging, and further chemical analysis is necessary to precisely determine their structures and the potential for novel compound discovery. In conclusion, this type of research is crucial to maintain hope in finding new antibiotics that can combat the rapidly increasing percentages and types of antibiotic-resistant strains. These strains pose a significant threat to human life, and it is essential to continue the search for novel compounds to address this pressing issue.