Mycoviruses are viruses that infect fungi and have the potential to control fungal diseases of crops when associated with hypovirulence. Typically, mycoviruses have double-stranded (ds) or single-stranded (ss) RNA genomes. No mycoviruses with DNA genomes have previously been reported. Here, we describe a hypovirulence-associated circular ssDNA mycovirus from the plant pathogenic fungus Sclerotinia sclerotiorum. The genome of this ssDNA virus, named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), is 2166 nt, coding for a replication initiation protein (Rep) and a coat protein (CP). Although phylogenetic analysis of Rep showed that SsHADV-1 is related to geminiviruses, it is notably distinct from geminiviruses both in genome organization and particle morphology. Polyethylene glycol-mediated transfection of fungal protoplasts was successful with either purified SsHADV-1 particles or viral DNA isolated directly from infected mycelium. The discovery of an ssDNA mycovirus enhances the potential of exploring fungal viruses as valuable tools for molecular manipulation of fungi and for plant disease control and expands our knowledge of global virus ecology and evolution.