Rosellinia necatrixis a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources forR. necatrixhas complicated a thorough understanding of its infection biology. Here, we sequenced nineR. necatrixstrains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, theR. necatrixgenome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome ofR. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists ofR. necatrixgrowthin vitroand can alleviateR. necatrixinfection on cotton plants. Collectively, our data show thatR. necatrixencodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.