A total of 103 isolates of basidiomycetes, representing 84 species from different Brazilian ecosystems, were evaluated for their antifungal and antibacterial activity in a panel of pathogenic and non-pathogenic microorganisms. Tissue plugs of the fruiting bodies were cultivated in liquid media and the whole culture extracted with ethyl acetate. Crude extracts from
Agaricus
cf.
nigrecentulus,
Agrocybe perfecta
,
Climacodon pulcherrimus
,
Gloeoporus thelephoroides
,
Hexagonia hydnoides
,
Irpex lacteus
,
Leucoagaricus
cf.
cinereus,
Marasmius cf.
bellus,
Marasmius
sp.,
Nothopanus hygrophanus
,
Oudemansiella canarii
,
Pycnoporus sanguineus
,
Phellinus
sp., and
Tyromyces duracinus
presented significant activity against one or more of the target microorganisms. Eight isolates were active only against bacteria while three inhibited exclusively the growth of fungi. Two extracts presented wide antimicrobial spectrum and were active against both fungi and bacteria. Differences in the bioactivity of extracts obtained from isolates from the same species were observed.