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Rock varnish is a darkly pigmented coating rich in manganese oxides. Though microbes inhabit varnish deposits, it is unclear whether they are involved in varnish formation. The fungal communities of rock varnish and adjacent rock sites with no visible varnish deposits were examined. Microcolonial fungi were identified at all sampling sites, and were associated with manganese oxides in patches of incipient varnish at non-varnish sites. Fungi were closely related to manganese-oxidizing genera and seventeen isolates oxidized manganese in culture, producing six distinct manganese-oxide morphologies. Our results indicate that microcolonial fungi may play a crucial role in rock varnish formation. Supplemental materials are available for this article. Go to the publisher’s online edition of Geomicrobiology Journal to view the free supplemental file.