Plant growth-promoting bacteria (PGPB) have gained significant attention for their role in enhancing plant growth and health through various mechanisms, including the production of phytohormones and nutrient solubilization. Of these beneficial microbes, Pseudomonas species have been widely recognized for their potential to stimulate plant growth and confer resistance to various biotic and abiotic stressors. To better understand the genetic basis of these plant–microbe interactions, it is essential to isolate high-quality genomic DNA from Pseudomonas species. In this context, we describe a robust protocol for the isolation of genomic DNA from Pseudomonas species, focusing on the optimization of extraction methods for PGPB strains. The isolation protocol combines a mechanical cell disruption step with chemical lysis, followed by DNA isolation by phenol–chloroform–isoamyl alcohol method. The efficiency of this protocol was assessed by measuring DNA yield and quality through gel electrophoresis. Additionally, the integrity of the extracted DNA was confirmed by polymerase chain reaction (PCR) amplification of housekeeping genes specific to Pseudomonas species. This method can facilitate research on the genetic basis of plant growth promotion by Pseudomonas species and enhance our understanding of the mechanisms underlying these beneficial interactions. Furthermore, this protocol can be adapted for the isolation of DNA from other microbial species, contributing to the broader field of microbiology and microbial ecology.