We address the concerns that managed forest plantations may cause reductions of diversity of understory vegetation. We performed inventories of two mature ponderosa pine plantations in which multiple treatments were applied during plantation establishment. At stand age 35, we measured tree heights and diameters, understory plant cover and diversity, soil nutrients and chemical properties, and soil microbial biomass and diversity with phospholipid fatty acid. We found a significant, positive effect from both herbicide (H) and fertilizer (F) applications on subsequent overstory tree growth and development (P < 0.05); insecticide (I) effect was minimal. We observed negative effects on understory plant diversity decades later when herbicide was applied during stand establishment. However, lower plant diversity and ground cover appeared to have been caused primarily from overstory canopy closure, supported by the increased understory cover and diversity observed in the HI and HFI plots that had been thinned at age 12. Similarly, while fertilizer increased tree growth at both sites, it only negatively affected understory plants at the higher quality site. We did not find significant influence of treatment effects on soil nutrients and microbial communities. Therefore, to mitigate the potential loss of understory biodiversity in plantations, foresters can manage overstory trees with traditional pre-commercial thinning techniques and early tending.