Microbial interactions in agricultural soils can play important roles in the control of soil-borne phytopathogenic diseases. Yields from blueberry plantations from southern Spain have been impacted by the pathogenic fungus, Macrophomina phaseolina. The use of chemical fungicides has been the common method for preventing fungal infections, but due to their high environmental impact, legislation is increasingly restricting its use. Biocontrol alternatives based on the use of microorganisms is becoming increasingly important. Using the metabarcoding technique, fungi and bacteria were characterized (via 16S and ITS regions, respectively) from rhizosphere soils of healthy and dead blueberry plants infected by M. phaseolina, and which had undergone three different treatments: two biocontrol strategies—one of them a mix of Pseudomonas aeruginosa and Bacillus velezensis and the other one with Bacillus amyloliquefaciens—and a third treatment consisting of the application of a nutrient solution. The treatments produced changes in the bacterial microbiota and, to a lesser extent, in the fungi. The abundance of Fusarium was correlated with dead plants, likely favoring the infection by M. phaseolina. The presence of other microorganisms in the soil, such as the fungi Archaeorhizomyces or the bacteria Actinospica, were correlated with healthy plants and could promote their survival. The different genera detected between dead and healthy plants opens the possibility of studying new targets that can act against infection and identify potential microorganisms that can be used in biocontrol strategies. View Full-Text ►▼ Show Figures This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.