To prevent infection by the root rot fungi Heterobasidion spp., surfaces of freshly cut Picea abies stumps are sprayed with Rotstop biocontrol agent, which constitutes a spore suspension of a single genotype of the competitive fungus Phlebiopsis gigantea. In Fennoscandia, the agent is applied on 47,000 ha of forestland yearly. The aims of the present work were to estimate the persistence of Rotstop P. gigantea strain and its impact on mycodiversity in 4- to 6-year-old P. abies stumps. Fungal isolation was done from 947 wood samples taken from 130 stumps, 60 of which were Rotstop-treated and 70 were untreated controls. Half of the stumps were 4-year, and another half 6-year-old. 705 fungal strains representing 81 species were isolated. Species richness was lower in Rotstop-treated stumps, both after 4 (by 32%; P = 0.08) and after 6 years (46%; P = 0.01). The majority of species (65–75%) isolated from Rotstop-treated stumps were also found in controls. In 4-year-old Rotstop-treated stumps, the fungal community was dominated by P. gigantea, both in terms of isolated strains and observed fruitbodies. Of P. gigantea isolates, only Rotstop genotypes were found in treated stumps, while control stumps harboured wild strains of the species. In 6-year-old both treated and control stumps, P. gigantea was seldom detected and no fruitbodies were observed. Here, the occurrence of other fungi increased, e.g., Resinicium bicolor, Sistotrema brinkmannii, and Hypholoma capnoides. The pathogen, Heterobasidion spp., was the only basidiomycete with a significantly reduced incidence in Rotstop-treated stumps.