Mycodnaviridae is a clade of giant viruses that persistently infect zoosporic fungi.

Giant viruses of the phylum Nucleocytoviricota have emerged as particularly notable due to their increasingly recognized impacts on eukaryotic genome evolution. Their origins are hypothesized to predate or coincide with the diversification of eukaryotes, and they have been detected in hosts that span the eukaryotic tree of life. But surprisingly, such viruses have not been definitively found in Kingdom Fungi, though earlier genomic and metagenomic work suggests putative associations. Here we report both "viral fossils" and active infection by giant viruses in fungi, particularly in the zoosporic phyla Blastocladiomycota and Chytridiomycota. The recovered viral assemblies span up to 350 kb, encode over 300 genes, and form a monophyletic family-level clade within the Nucleocytoviricota related to orders Imitervirales and Algavirales, which we name Mycodnaviridae. We observed variation in infection status among the isolates including apparent active infection and transcriptionally suppressed states, suggesting that viral activation may be constrained to certain life stages of the host. Our experimental findings add to the limited natural virus-host systems available in culture for the study of giant viruses and expand the known host range of Nucleocytoviricota into a new kingdom that contains many model species. Mycodnaviridae have a global distribution, which invites inquiry into the implications of these infections for host traits, host genome evolution, and the metabolic impacts on ecosystems.