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Abstract Background The global consumption of herbal medicine is increasing steadily, posing an extinction risk to medicinal plants. Uganda is among the top ten countries with a high threat of herbal medicine extinction, and Traditional Medicinal Knowledge (TMK) erosion. This might be attributed to the inadequate documentation, plus many more unclear hindrances. In this study, plant species used to treat human diseases in Butaleja district in Eastern Uganda and their associated TMK were documented. The conservation methods for medicinal plants were also evaluated. The rationale was to support the preservation of ethnopharmacological knowledge. Methods Data were collected from 80 herbalists using semi-structured questionnaires, from July 2020 to March 2021. Additionally, guided field walks and observations were conducted. Quantitative indices such as, use categories and informant consensus factor (ICF) were evaluated to elucidate the importance of the medicinal plants. Data were analyzed using STATA version-15.0 software. Results In total, 133 species, belonging to 34 families and 125 genera were identified. Fabaceae (65%), and Solanaceae (29%) were the dominant families. Leaves (80%), and roots (15%), were the commonest parts used in medicinal preparations; mostly administered orally as decoctions (34.6%) and infusions (16%). The commonest illnesses treated were cough (7.74%), gastric ulcers (7.42%), and malaria (4.52%). The informant consensus factor was high for all disease categories (≥ 0.8), indicating homogeneity of knowledge about remedies used. Only 73% of the respondents made efforts to conserve medicinal plants. The commonest conservation strategy was preservation of forests with spiritually valued species (100%), while compliance with government regulations was the rarest (4.5%). Overall, efforts to stop the extinction of medicinal plants and TMK were inadequate. Conclusion and recommendations There was enormous dependency on a rich diversity of medicinal plant species and TMK for healthcare and income generation. The potential for medicinal plant biodiversity loss was evident due to habitat destruction. Inclusion of traditional cultural norms in conservation strategies, and laboratory-based efficacy tests for the species identified are necessary, to promote the conservative and utilization of validated herbal medicines and TMK in rural settings.