Evaluation of the in vitro antibacterial activity of Methanolic extracts isolated from Henna (Lawsonia inermis) leaf against two nosocomial infection causing pathogens, gram-positive Staphylococcus aureus and gram-negative Escherichia coli. This interventional study was carried out during the period of January 2021 to December 2021 in the Department of Pharmacology and Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. The antibacterial activity was tested at different concentrations of Methanolic Henna leaf extracts by using disc diffusion and broth dilution method. The extract was prepared by using solvents Methanol and 0.1% DMSO (Dimethyl sulfoxide). The test microorganisms were also tested for their activity against a standard antibiotic Ciprofloxacin by broth dilution method and the result was compared with that of Methanolic leaf extracts. Methanolic Henna leaf Extracts (MHE) were used initially in nine different concentrations (2.5, 5, 10, 20, 50, 100, 200, 500 and 1000 mg/ml) and later in selected concentrations as needed to confirm the more precise margin of antimicrobial sensitivity of the extracts. Among different concentrations of the MHE, 100mg/ml and above concentrations showed inhibitory effect against aforesaid bacteria. The MIC for Staphylococcus aureus and Escherichia coli were 100 and 200 mg/ml in MHE respectively. The MIC of Ciprofloxacin was 1μg/ml against both Staphylococcus aureus and Escherichia coli. The MIC of Ciprofloxacin was the lowest in comparison to MICs of MHE for the test organisms. The present study showed that Methanol Henna extracts demonstrated antibacterial effects against nosocomial infection pathogens. From this study, it is clearly observed that there are definite antibacterial effects of the methanolic extract of Henna leaves (Lawsonia inermis) against Staphylococcus aureus and Escherichia coli.