The sympatric occurrence of species is thought to be based mainly on the differences in their use of habitat and of limiting resources. Segregating parameters may be of spatial or temporal character and may include behavioral differences. We hypothesized that species of large hunting spider living sympatrically in a Costa Rican lowland rain forest should differ in their habitat and/or hunting microhabitat preferences, in daily activity pattern, and, as an adaptation to the preferred hunting microhabitat, in their specific ability to adhere to smooth surfaces. We found an assemblage of eight large species of the families Ctenidae and Trechaleidae, consisting of three subguilds: 1) two semi-aquatic species with low adhesion ability, 2) three forest-floor dwelling species with good adhesion ability, and 3) three vegetation dwelling species showing very good adhesion ability. The species were partially segregated by habitat type, with two of the vegetation dwelling species preferring the treeless area of a temporary swamp. We found no species-specific differences in daily activity patterns. The similarity in community structure between this Costa Rican and a central Amazonian assemblage suggests the existence of similar structuring mechanisms in wandering spider assemblages in climatically similar biomes.