Due to the emergence of drug-resistant microorganisms, the search for broad-spectrum antimicrobial compounds has become extremely crucial. Natural sources like plants and soils have been explored for diverse metabolites with antimicrobial properties. This study aimed to identify microorganisms from agricultural soils exhibiting antimicrobial effects against known human pathogens, and to highlight the chemical space of the responsible compounds through the computational metabolomics-based bioprospecting approach. Herein, bacteria were extracted from soil samples and their antimicrobial potential was measured via the agar well diffusion method. Methanolic extracts from the active bacteria were analyzed using the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technique, and the subsequent data was further analyzed through molecular networking approach which aided in identification of potential anti-microbial compounds. Furthermore, 16S rRNA gene sequencing enabled identification of the active bacterial isolates, where isolate 1 and 2 were identified as strains of Bacillus pumilus, whilst isolate 3 was found to be Bacillus subtilis. Interestingly, isolate 3 (Bacillus subtilis) displayed wide-ranging antimicrobial activity against the tested human pathogens. Molecular networking revealed the presence of Diketopiperazine compounds such as cyclo (D-Pro-D-Leu), cyclo (L-Tyr-L-Pro), cyclo (L-Pro-D-Phe), and cyclo (L-Pro-L-Val), alongside Surfactin C, Surfactin B, Pumilacidin E, and Isarrin D in the Bacillus strains as the main anti-microbial compounds. The application of the molecular networking approach represents an innovation in the field of bio-guided bioprospection of microorganisms and has proved to be an effective and feasible towards unearthing potent antimicrobial compounds. Additionally, the (computational metabolomics-based) approach accelerates the discovery of bioactive compounds and isolation of strains which offer a promising avenue for discovering new clinical antimicrobials. Finally, soil microbial flora could serve an alternative source of anti-microbial compounds which can assist in the fight against emergence of multi-drug resistance bacterial pathogens.