Abstract Pleurotus ostreatus (oyster mushroom) is a globally cultivated and highly nutritious mushroom. However, viral infections posed significant challenges to its cultivation, causing tremendous loss of quality and yield. The fruiting bodies with the symptom of lace-like malformation were observed in a farm from Fangshan district, Beijing. To explore the possible associated virus(es), P. ostreatus was purified from a single fruiting body and the mycelium collected for ribosomal RNA-depleted total RNA sequencing. Eight RNA mycoviruses were identified through bioinformatic analysis and experimental verification, including three with double-stranded RNA (dsRNA) genomes in the Partitiviridae family, two with circular RNA genomes in the families of Quambiviridae and Dumbiviridae , one with negative-sense single-stranded RNA (−ssRNA) genome in the family Mymonaviridae , and two with positive-sense single-stranded RNA (+ssRNA) genomes (one in Deltaflexiviridae and one unclassified). The characteristics of these viruses, such as genome structure, conserved domains and their phylogenetic relationships with known viruses were further determined and seven were demonstrated to be novel viruses. These findings enriched our understanding of the virus diversity in P. ostreatus , providing valuable information for further research on the interaction between the viruses and the host mushroom to improve its cultivation.