Ethno botany is a broad term referring to the study of relationship between people and plants. The main objective of this study is to conduct an ethnobotanical study of medicinal plants and antibacterial test of selected medicinal plant extracts in Sedie Muja District. This research was conducted in Sedie Muja District, South Gondar Administration Zone of Amhara Regional State, and Northwest Ethiopia. Ethnobotanical data were collected from January 2025 to April 2025. A total of 96 informants from the four study kebeles (78 general informants and 18 key informants) were selected. Ethnobotanical data were gathered through unstructured interview, group discussion, guided field walk and market survey. The collected data were analyzed using preference ranking, direct matrix ranking, informant consensus factor, fidelity level index and one way ANOVA at 95% confidence level between means using SPSS software version 21. A total of 76 medicinal plant species in 70 genera and 45 families were recorded. From those, 39 species (51%) were cited for the treatment of only human ailments whereas 13(17%) species were reported for treating only livestock ailments and 24 species(32%) for treating both humans and livestock ailments. Shrubs constituted the largest growth habit (33species) followed by herbs (26 species). Forty (40) human ailments in ten disease categories were identified. Among these, animal biting (0.89) diseases categories had the highest ICF values. Seventeen veterinary (17) ailments in 8 disease categories were identified. Few medicinal plants were sold in the market. Fuel wood collection, agricultural land expansion, overgrazing, construction, and timber production, were the threatening factors of medicinal plants and associated indigenous knowledge. Four medicinal plants (Ostostegia integrifolia, Clematis simnesis, Verbascum sinaiticum and Utrica simensis were investigated for their antibacterial properties using agar disk diffusion method using methanol solvents. The highest antibacterial activity (13.33 mm) was observed from methanol extract of Verbascum sinaiticum root extract. The minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) was recorded in Verbascum sinaiticum root extract. In the present study, 76 plant species of medicinal importance were recorded and documented. In the district 40 human disease types and about (17) veterinary ailment were identified. Threats on medicinal plants in the study area were agricultural expansion, charcoal making and fire wood collection, collection of construction woods, overgrazing, timber production and cutting. Further studies should be conducted on qualitative and quantitative phytochemical analysis on the medicinal plants such as Verbascum sinaiticum and Clematis simensis that do have better antibacterial activity. Key word : Antibacterial activity, Indigenous Knowledge and Informant consensus factor