Vernonia amygdalina belongs to the family Asteraceae. Ethiopia and other tropical parts of Africa are home to the plant. The present investigation examined the antibacterial properties, separation, and structural elucidation of the root extract of Vernonia amygdalina. Using methanol and chloroform as solvents, the plant's powdered and air-dried roots were extracted via maceration. Disc diffusion antibacterial tests were performed with the crude methanol and chloroform extracts at concentrations of 25, 50, 75 and 100 mg/mL and Salmonella typhi (S. typhi), Escherichia coli (E. coli), Streptococcus pneumonia (S. pneumonia) and Staphylococcus aureus (S. aureus) being the tested bacteria. The chloroform extract of the plant root was found to inhibit all cultures of the four bacteria, while the methanol extract inhibited cultures of only three bacteria; S. aureus was thereby not inhibited. However, the two extracts showed a lower inhibition activity than standard antibiotics. The most active crude extract, the methanol extract, was subjected to phytochemical tests and structural characterization. It was found to contain alkaloids, tannins, flavonoids, terpenoids and saponins. Structural elucidation using 1 H- NMR, (13) C-NMR, Dept-135 NMR and IR spectroscopy was applied to identify the compound VA-14 in the methanol extract.