Ethnobotany is a broad term referring to the study of relationship between people with Plants. The main objective of this study is to conduct an ethnobotanical study of medicinal plants and antibacterial test of selected medicinal plants in Dera District. This research was conducted in Dera District, South Gondar Administration Zone of Amhara Regional State, Ethiopia. Ethnobotanical data were collected from November, 2020 to January, 2020. A total of 120 informants from four study kebeles (100 general and 20 key informants) were selected. The ethnobotanical data were gathered through semi-structured interview, Group discussion, guided field walk and market survey. A total of 74 medicinal plant species in 69 genera and 44 families were recorded. From those, 37 species were cited for the treatment of only human ailments whereas 13 species were reported for treating only livestock ailments and 24 species for treating both humans and livestock ailments. Thirty nine (39) human ailments in ten disease categories were identified. Among these, animal biting (0.89) diseases categories had the highest ICF values. Eighteen veterinary 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 (Otostegia integrifolia, Clematis simensis, Verbasicum sinaiticum and Girardinia bullosa were investigated for their antibacterial properties using agar disk diffusion method using methanol solvents. The highest antibacterial activity (12.33 mm) was observed from methanol extract of Verbasicum sinaiticum root extract. The minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) was recorded in Verbasicum sinaiticum root extract. Key Word / Phrase: Antibacterial activity, Indigenous Knowledge, Threatining factor.