The need to screen efficient, safe, affordable, and readily available antimicrobial agents from various medicinal plants, for their potential antimicrobial activity has become critical in light of the rapid development of microbial resistance against chemotherapeutic drugs (mainly antibiotics). Despite notable advances in the biological and phytochemical evaluation in recent years, comprehensive reviews of Holarrhena pubescens are limited. The main objective of this study is to evaluate the antimicrobial activities of this plant extract. One significant use of plant extracts can be in treating bacterial infections and develop potential antibacterial agents against multi drug resistant (MDR) and susceptible bacteria. If antimicrobial activities are present, these extracts will provide useful information for discovering new compounds with better activity and more effectiveness against resistant and susceptible bacteria responsible for infections than currently available. The ethanolic extract of leaves of Holarrhena pubescens was prepared and tested against multidrug resistant (MDR) and American type culture collection strains of bacteria. We have obtained two types of results in this experiment. The extract showed antimicrobial properties against Staphylococcus aureus (ATCC 23235), Escherichia coli (MDR) and Acinetobacter (MDR), while, it showed no antimicrobial activities along with some growth promoting role against Escherichia coli (ATCC 25922), and Klebsialla pneumoniae (MDR), which may be due to chemicals other than secondary metabolites.