Habitat loss, fragmentation, and invasive species are major threats to biodiversity. In the Lower Rio Grande Valley (LRGV) of southern Texas, a conservation hotspot, few studies have examined how land use change and biotic disturbance influence biodiversity, particularly among Lepidoptera. We surveyed 24 habitat fragments on private lands in the LRGV and examined how patch size, edge to interior ratio (EIR), prevalence of invasive, exotic, and pest (IEP) plant species, and other environmental factors influenced plant and Lepidoptera communities within four habitat classes. Biotic disturbance was widespread and intense. IEP plants represented three of the four most common species in all but one habitat class; yet, classes largely had distinctive plant and Lepidoptera communities. Larger habitat patches had lower IEP prevalence but also lower plant richness and lower Lepidoptera richness and abundance. Conversely, patches with higher EIRs had greater IEP prevalence, plant richness, and Lepidoptera richness and abundance. IEP prevalence was negatively related to plant diversity and positively related to woody dominance, blooming plant abundance, and, surprisingly, both plant cover and richness. However, plant richness, abundance, and diversity were higher where a greater proportion of the plants were native. Lepidoptera diversity increased with plant cover, and Lepidoptera richness and abundance increased with plant richness. More individual Lepidoptera species were influenced by habitat attributes than by availability of resources such as host plants or nectar sources. Our results illustrate extensive landscape alteration and biotic disturbance and suggest that most regional habitats are at early successional stages and populated by a novel species pool heavy in IEP species; these factors must be considered together to develop effective and realistic management plans for the LRGV.