Biocontrol agents reared in the laboratory or produced for commercial purposes may show deterioration in important biocontrol traits over time, reducing their field efficacy. To understand deterioration, we characterized phenotypic changes in biocontrol traits of five replicate lines each for two entomopathogenic nematode species and their associated bacteria subcultured repeatedly under standard lab culture procedures. Bacterial cultures showed changes in color and pH, and cells and inclusion bodies became smaller. One or more, and in some cases all five, nematode replicate lines showed declines in virulence, reproduction, heat tolerance, host seeking ability, and nictation. Heterorhabditis bacteriophora lines deteriorated faster and more extensively than those of Steinernema carpocapsae. Genetic analysis will reveal whether the mechanism for these losses is genetically (inbreeding, genetic drift, or inadvertent selection) or non-genetically based (e.g., nutrition, disease).