Stand tending using glyphosate to promote coniferous overstory trees has been a common practice in the boreal forests of Alberta. However, there are concerns about the impact of this practice on biodiversity of understory species. This study examined the impact of broadcast glyphosate application during the active reforestation phase, two to several years post-harvest, on forest plant diversity 25 to 40 years post-harvest. Herbicide treatments had the desired effect of shifting tree layer dominance from deciduous to coniferous species, driven by a 25-fold reduction in the density of trembling aspen (3927 vs. 154 stems·ha−1, untreated and treated, respectively). However, understory plant diversity was not significantly different between treated and untreated sites as examined by the Shannon–Wiener (H) and evenness (E) indices. Shared plant species (beta) across sites was high. Of the seven site-indicator species examined, three had significantly lower cover on treated sites: Wild sarsaparilla, low bush-cranberry and oak fern. Total understory plant cover was significantly greater in the treated portion (98.0%) versus untreated (71.4%); however, this difference was not significant when bryophytes were excluded in the analysis. The establishment, maintenance and monitoring of larger long-term trials is strongly recommended.