Background <p>The trans-kingdom movement of microRNAs (miRNAs) is a key regulatory mechanism in plant biotic interactions, influencing and modulating both pathogenic virulence and symbiotic relationships. This study employed bioinformatic analysis to predict the role of rice (<i>Oryza sativa</i> L.) miRNAs in regulating gene expression during interactions with the pathogenic fungi <i>Rhizoctonia solani</i> (<i>R. solani</i>) and <i>Magnaporthe oryzae</i> (<i>M. oryzae</i>), as well as the symbiotic fungus <i>Rhizophagus irregularis</i> (<i>R. irregularis</i>).</p> Results <p>Previously reported and up-regulated rice miRNAs were identified from the literature during inoculation, and their target genes were predicted in the fungal transcriptomes. The KEGG pathway and GO enrichment analyses revealed that rice miRNAs could target genes crucial for pathogen metabolism (e.g., carbohydrate, amino acid, lipid, and xenobiotic), genetic information processing, and essential cellular processes (e.g., signal transduction, transport, catabolism, and cell growth and death), potentially impairing fungal virulence and survival. The osa-miR171 family is predicted to regulate <i>R. irregularis</i> genes involved in protein serine/threonine kinase activity, antiporter activity, cell division, TAP complex binding, and O-acetylhomoserine sulfhydrylase activity during symbiosis. Additionally, the osa-miR171 family targets key rice genes such as phosphate transporter, NSP2, and SCR, involved in nutrient transport, common symbiosis signaling, and root development, respectively, which are likely important for forming and regulating a symbiotic relationship. The qRT‒PCR results confirmed the up-regulation of osa-miR171h (symbiosis-related) and osa-miR167d-5p (pathogen-responsive) in rice roots inoculated with <i>R. irregularis</i>.</p> Conclusions <p>These findings highlight the significant role of trans-kingdom RNA regulation in plant–microbe interactions, contributing to understanding the molecular regulation, distinctions, and similarities between pathogenic and symbiotic relationships in plants. This initial study performs the first comparative trans-kingdom sRNA analysis in rice, successfully predicting and distinguishing the molecular mechanisms that drive virulence versus mutualism. This work also lays the groundwork for developing novel strategies to enhance plant disease resistance, foster beneficial symbiotic interactions, and ultimately boost agricultural yields.</p>