Biofertilizers supply living microorganisms to help plants grow and keep their health. This study examines the microbiome composition of a commercial biofertilizer known for its plant growth-promoting activity. Using ITS and 16S rRNA gene sequence analyses, we describe the microbial communities of a biofertilizer, with 163 fungal species and 485 bacterial genera found. The biofertilizer contains a variety of microorganisms previously reported to enhance nutrient uptake, phytohormone production, stress tolerance, and pathogen resistance in plants. Plant roots created a microenvironment that boosted bacterial diversity but filtered fungal communities. Notably, preserving the fungal-inoculated substrate proves critical for keeping fungal diversity in the root fraction. We described that bacteria were more diverse in the rhizosphere than in the substrate. In contrast, root-associated fungi were less diverse than the substrate ones. We propose using plant roots as bioreactors to sustain dynamic environments that promote the proliferation of microorganisms with biofertilizer potential. The study suggests that bacteria grow close to plant roots, while root-associated fungi may be a subset of the substrate fungi. These findings show that the composition of the biofertilizer may be influenced by the selection of microorganisms associated with plant roots, which could have implications for the effectiveness of the biofertilizer in promoting plant growth. In conclusion, our study sheds light on the intricate interplay between plant roots and the biofertilizer's microbial communities. Understanding this relationship can aid in optimizing biofertilizer production and application, contributing to sustainable agricultural practices and improved crop yields.