Basidiomycetous macrofungi have therapeutic potential due to antimicrobial activity but little information is available for Australian macrofungi. Therefore, the present study investigated 12 Australian basidiomycetous macrofungi, previously shown to have promising activity againstStaphylococcus aureusandEscherichia coli, for their antimicrobial potential against a range of other clinically relevant micro-organisms. Fruiting bodies were collected from across Queensland, Australia, freeze-dried and sequentially extracted with water and ethanol. The crude extracts were tested at 10 mg/mL and 1 mg/mL against six pathogens including two Gram-positive and two Gram-negative bacteria along with two fungi using a high throughput 96-well microplate bioassay. A degree of specificity in activity was exhibited by the water extract ofRamariasp. (Gomphaceae) and the ethanol extracts ofPsathyrellasp. (Psathyrellaceae) andHohenbueheliasp., which inhibited the growth of the two fungal pathogens used in the assay. Similarly, the ethanol extract ofFomitopsis lilacinogilva(Fomitopsidaceae) was active against the Gram-positive bacteriaB. cereusonly. Activity against a wider range of the microorganisms used in the assay was exhibited by the ethanol extract ofRamariasp. and the water extract ofHohenbueheliasp. (Pleurotaceae). These macrofungi can serve as new sources for the discovery and development of much needed new antimicrobials. Copyright © 2011 John Wiley & Sons, Ltd.