A new ascomycete species, Natipusilla bellaspora, collected from submerged woody debris in a freshwater stream at Los Amigos Biological Station, Madre De Dios in the Peruvian Amazon is described and illustrated. This fungus is characterized by small, globose to subglobose, hyaline ascomata; small, globose to subglobose, eight-spored fissitunicate asci; one-septate, multiguttulate ascospores with two different gelatinous sheaths, an outer amorphous sheath that enlarges in water and an inner sheath that has a distinctive persistent shape and is attached to the ascospore apex. Morphologically N. bellaspora differs from other Natipusilla species in having larger ascospores and two ascospore sheaths. A second Natipusilla species, N. limonensis, is reported for the first time from Peru. Based on the unique morphological characters of taxa in Natipusilla and results of previous molecular phylogenetic analyses with other members of the Dothideomycetes, we establish Natipusillaceae fam. nov. for this unique tropical freshwater clade.