Plant growth-promoting rhizobacteria (PGPR) are increasingly applied to enhance crop productivity and support sustainable agriculture. Among them, Pseudomonas spp. have demonstrated multiple beneficial traits, including nutrient mobilization, phytohormone production, and suppression of soilborne pathogens. However, there is growing evidence that the introduction of PGPR may also impact native soil microbial communities by altering their composition, diversity, and functional interactions. In this context, the aim of the study (part of project: PRIN 2022LPPFTY, -TREASURE) was to analyze the bacterial community assemblage of soil, after inoculation with Pseudomonas extremaustralis. To this end, three bacterial concentrations (10⁶, 10⁸, and 10¹⁰ CFU) and three inoculation methods were tested on soils on which Hordeum vulgare L. (barley) plants were grown. The inoculation approaches included: soaking seeds in bacterial suspensions prior to sowing (SEED), immersing roots of pre-germinated seeds in bacterial solutions before planting (ROOT) and irrigating the soil with bacterial suspensions after planting pre-germinated seeds (SOIL). Control treatments followed the same procedures but used sterile distilled water in place of bacterial solutions. Each treatment was replicated five times. At the end of the experiment (2 months), DNA was extracted from soil and the 16S gene regions were sequenced. The results highlighted that, regardless of the inoculation methods, the bacterial concentration impacted on the bacterial communities, as the soil with 10¹⁰ bacterial CFU showed higher richness and Shannon index, than the others. However, the inoculation method also had an impact on soil bacterial community, as they showed significant difference in bacteria assemblage, in particular in the soil inoculated with ROOT method. This study demonstrates that PGPR application can modify soil bacterial community structure, highlighting the importance of optimizing both the dose and method of application. This is fundamental to balance plant benefits with potential shifts in native soil microbial communities, thus supporting more sustainable agricultural practices.