A broad range of environmental conditions likely regulate predator–prey population dynamics and impact the structure of these communities. Central to understanding the interplay between predator and prey populations and their importance is characterizing the corresponding trophic interactions. Here, we use a well-documented molecular approach to examine the structure of the community of natural enemies preying upon the squash bug, Anasa tristis, a herbivorous cucurbit pest that severely hinders organic squash and pumpkin production in the United States. Primer pairs were designed to examine the effects of organic management practices on the strength of these trophic connections and link this metric to measures of the arthropod predator complex density and diversity within an experimental open-field context. Replicated plots of butternut squash were randomly assigned to three treatments and were sampled throughout a growing season. Row-cover treatments had significant negative effects on squash bug and predator communities. In total, 640 predators were tested for squash bug molecular gut-content, of which 11% were found to have preyed on squash bugs, but predation varied over the season between predator groups (coccinellids, geocorids, nabids, web-building spiders and hunting spiders). Through the linking of molecular gut-content analysis to changes in diversity and abundance, these data delineate the complexity of interaction pathways on a pest that limits the profitability of organic squash production.