Pathogens resistant to antimicrobials form a significant threat to public health worldwide. Tackling multidrug-resistant pathogens via screening metagenomic libraries has become a common approach for the discovery of new antibiotics from uncultured microorganisms. This study focuses on capturing nonribosomal peptide synthase (NRPS) gene clusters implicated in the synthesis of many natural compounds of industrial relevance. A NRPS PCR assay was used to screen 2976Escherichia coliclones in a soil metagenomic library to target NRPS genes. DNA extracts from 4 clones were sequenced and subjected to bioinformatic analysis to identify NRPS domains, their phylogeny, and substrate specificity.Successfully, 17 NRPS-positive hits with a biosynthetic potential were identified. DNA sequencing and BLAST analysis confirmed that NRPS protein sequences shared similarities with members of the genusDelftiain theProteobacteriataxonomic position. Multiple alignment and phylogenetic analysis demonstrated that clones no. 15cd35 and 15cd37 shared low bootstrap values (54%) and were distantly far from close phylogenetic neighbors. Additionally, NRPS domain substrate specificity has no hits with the known ones; hence, they are more likely to use different substrates to produce new diverse antimicrobials. Further analysis confirmed that the NRPS hits resemble several transposon elements from other bacterial taxa, confirming its diversity. We confirmed that the analyses of the soil metagenomic library revealed a diverse set of NRPS related to the genusDelftia.An in-depth understanding of those positive NRPS hits is a crucial step for genetic manipulation of NRPS, shedding light on alternative novel antimicrobial compounds that can be used in drug discovery and hence supports the pharmaceutical sector.