Vitex negundo is an aromatic shrub distributed worldwide with several medicinal properties. The alarming and rapid increase in the antimicrobial drug resistance globally has led to the development of alternative therapeutic strategies to overcome drug resistance. Hence, the present study was carried out to evaluate the antibacterial efficacy of the acetone, benzene, ethyl acetate, chloroform, and methanolic leaf extracts of V. negundo against Klebsiella pneumonia (K. pneumonia) using in vitro studies. The sensitivity test against the standard antibiotics showed that the tested K.
pneumoniae was multi drug resistant (MDR) bacteria. Based on the results of the Thin Layer Chromatography (TLC) bioassay, ethyl acetate was selected as the suitable solvent for extraction of V. negundo leaves. The zone of inhibition (ZoI) was observed starting from the concentration of 50 μg/mL for the V. negundo ethyl acetate leaf extract for the selected MDR bacteria K. pneumoniae. The highest ZoI was observed as 18 mm at the concentration of 200 mg/mL. Ethyl acetate leaf extract of V. negundo also expressed MIC value of 1.5 μg/mL against the test pathogen MDR K. pneumoniae. The inhibitory effect of the ethyl acetate leaf extract of V. negundo leaves was apparent from 40 minutes indicating time dependant inhibition or killing of the MDR K. pneumoniae. The results of the present study demonstrate the antibacterial activity of the ethyl acetate leaf extract of V. negundo, which may be attributed to the presence of several secondary metabolites in the extract.