Two entomopathogenic nematodes, Steinernema colombiense sp. nov. (strain SNIO198) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (strain Fresno HNI0100) (Rhabditida: Heterorhabditidae), were isolated from the Colombian Andes and cultured in the laboratory. In semi-field tests against the ground pearl Eurhizococcus colombianus Jakubski (Hemiptera: Margarodidae) on blackberry, neither species was pathogenic to crawlers and only S. colombiense was moderately infective against adults at 105 IJ/ml. Higher activity for both nematodes was observed against immature spittlebugs Aeneolamia sp. (Hemiptera: Cercopidae) infesting kikuyu grass, with H. bacteriophora and S. colombiense killing 100% and 75% of insects, respectively, in our tests. Our results demonstrate the potential use of native strains of entomopathogenic nematodes as biological control agents in Colombia.