A simple and rapid procedure for efficiently isolating fungi DNA suitable for use as a template for PCR amplification and other molecular assays is described. The main advantages of the method are: (1) the mycelium is directly recovered from Petri-dish cultures; (2) the technique is rapid and relatively easy to perform , and (3) it allows for processing of around 50 samples during a single day; (4) it is inexpensive; (5) the quality and quantity of DNA obtained are suitable for molecular assays; (6) it can be applied to filamentous fungi from soil as well as from a fungi from other environmental sources; and (7) it does not require the use of expensive and specialized equipment or hazardous reagents.