Interrow cover crop implementation is considered a promising and sustainable agronomic practice for enhancing crop performance and soil health in agroecosystems, mediated by improvement of the soil microbiome. This study assessed how interrow cover crop implementation affected the microbial community in the soil, and may enhance its suppressiveness against Verticillium dahliae in a commercial olive orchard. The experiments were performed in a commercial olive orchard divided into two different management zones: conventional (trees without interrow cover crops treatment) and the LivinGro® protocol (trees with interrow cover crops treatment). Soil samples were collected focusing in 2 sampling times (September 2021 and January 2022). Soil DNA was extracted, and 16S rRNA genes and ITS regions sequences analyzed to profile soil microbial communities. Cover crop implementation does not increase microbial richness and alpha diversity values. However, we observed that the use of cover crops influences the composition of both fungal and bacterial soil microbial communities. Thus, cover crop implementation in the soil significantly increased the relative abundance of some putative beneficial bacterial groups, such as Bacillaceae, Blastocatellaceae and Koribacteraceae. In addition, compared with conventional soil, the soil treated with cover crops displayed increased suppressiveness against the olive soil-borne pathogen Verticillium dahliae.