To control a disease it is essential to know its causal agent. A correct diagnosis provides a considerable amount of basic information to select correct control methods. An accurate diagnosis always starts with a representative sample of the damaged tissue, from which a portion is taken, to isolate the causal agent. In the case of phytopathogenic fungi, there are several isolation methods, depending on the tissue or substrate in which they are found. This protocol describes the isolation and purification of phytopathogenic fungi from leaves and fruits of different tropical crops.