Plants have been used as medicine since time immemorial (Ushimaru et al., 2007). Medicinal plants are essential sources of easily accessible remedies used by traditional healers. Henna leaves are used to cure jaundice, skin diseases, dysentery, arthritis (Sharma et al., 2018). Lawsonia inermis is widely used by Rwandan as cosmetic products and in treatment of different ailments. This study was aimed to investigate the phytochemical screening and in vitro antimicrobial activities of different extracts of L. inermis barks collected from Huye District in Southern Province of Rwanda. The dried and powdered barks were extracted with methanol and cyclohexane by maceration giving 1.627g (10.83%), 0.173.g (1.15%) respectively. The extracts were concentrated for further phytochemical tests and evaluated for antimicrobial activity against Escherichia coli and Staphylococcus aureus using disk diffusion method.The results from phytochemical screening revealed the presence of terpenoids, phenolic compounds, tannins, flavonoids, proteins and saponins. Lawsonia inermis barks displayed antimicrobial activity against both gram negative and gram positive bacterial strains used in the present study. The findings of antimicrobial assay showed that the methanol extract of lawsonia inermis barks with the concentration of 10-1 has an antibacterial activity against gram negative bacteria Escherichia coli with zone of inhibition of 10 mm which is same as, the positive control, penicillin inhibition zone (10 mm) with the same concentration. The antibacterial activity of cyclohexane extract against E. coli showed a smaller inhibition zone of 9 mm for diluted inoculum (10-1). For the same concentrations of extracts and inoculums, the methanol extract inhibited Staphylococcus aureus (gram positive) growth with zone of inhibition of 9 mm, while the antibacterial activity of cyclohexane extract against the same bacteria was 4 mm, which are smaller than penicillin inhibition zone (18 mm). Key words: lawsonia inermis, phytochemical screening, anti-microbial activity, E. coli, S. aureus.