A laboratory assay to assess colonization of a submersed aquatic plant, Eurasian water milfoil (Myriophyllum spicatum), by fungi was developed and used to evaluate the colonization potential of Colletotrichum gloeosporioides, Acremonium curvulum, Cladosporium herbarum, Aureobasidium pullulans, a Paecilomyces sp., and an unidentified sterile, septate fungus. Stem segments of plants were first immersed in suspensions of fungal propagules for 24 h and then washed to remove all but the tightly attached component of the population. Inoculation was followed by two growth cycles of 3 days each. At the start of each cycle, washed plants were transferred to a mineral salts medium to provide an opportunity for the attached fungal populations to grow. After each growth period, plants were again washed, and fungal populations in the medium (nonattached), loosely attached and tightly attached to the plant, and within the plant (endophytic) were assayed by dilution plating. The fungi differed in the extent to which they attached to water milfoil and in their ability to grow in association with it. There were relatively few significant differences among the tightly attached fungal populations after 24 h, but growth of the better colonizers led to a greater number of significant differences after 4 and 7 days. In addition, the better colonizers showed sustained regrowth of loosely and nonattached fungal propagules in the face of intermittent removal by washing. A milfoil pathogen, C. gloeosporioides, was the only endophytic colonizer; it was also among the best epiphytic colonizers but was not demonstrably better than A. curvulum, a fungus commonly found as an epiphyte on watermilfoil. The yeastlike hyphomycete Aureobasidium pullulans was the only fungus that consistently failed to establish an increasing population on the plant.