The objective of this study was to assess the effects of forest application of diflubenzuron (Dimilin®), a gypsy moth suppressant, on nontarget invertebrates in litter and soil on the forest floor. Four experimental watersheds (two treatment and two reference) in the Fernow Experimental Forest, West Virginia, were sampled for two years before and one year after application of diflubenzuron (70 g ha−1, AI). Soil and leaf litter arthropods were monitored before and after application using soil cores and litter bag colonization samplers. Frequency analysis, BACIP, and ANOVA were used to analyse the data for diflubenzuron treatment effects. During the 36 month study, soil core samples were dominated by mites (49%) and springtails (28%). A total of 19 taxonomic groups were suitable for statistical analysis. We detected no significant treatment effects based on total organism counts or counts by trophic categories (p < 0.05). There were no significant treatment effects for populations of major taxonomic groups, except for Araneae (spiders). Analysis of leaf litter bags also showed no significant differences in total numbers of invertebrates or in trophic categories between treated and reference watersheds during the 12 month post-treatment study. Density of one species of springtail (Sminthurus purpurescens) and springtails as a group were significantly lower in treated watersheds. High variation was associated with both soil core and leaf bag samples; because of this variation, a significance level of 0.05 should be considered very conservative. Long-term trends in soil biota were evident, demonstrating the need for well-established pre-treatment baseline data for pesticide impacts on soil organisms