Basil is a popular medicinal and culinary herb, and its essential oils have been used extensively for many years in food products, perfumery, dental and oral products. Basil essential oils and their principal constituents were found to exhibit antimicrobial activity against a wide range of Gram-negative and Gram-positive bacteria, yeast, and mould. The essential oils obtained from aerial parts of three different species of Ocimum comprising twenty one germplasm lines were investigated for their essential oil composition and antimicrobial activity during 2010. Essential oils from seventeen germplasm lines in Ocimum basilicum and two each in Ocimum tenuiflorum and Ocimum gratissimum were investigated for anti-microbial activity against four bacterial strains (Staphylococcus aureus, Bacillus sps., Escherichia coli and Pseudomonas aeruginosa). The morpho-chemotypes exhibited wide variability for morphological and chemical traits. Anti-bacterial activity was found to be high for Staphylococcus aureus, moderate for Escherichia coli, low for Bacillus and Pseudomonas aeruginosa was highly resistant. The essential oils of Pale Green-Broad Leaves (O. basilicum) and CIM Ayu (O. gratissimum) exhibited significant antibacterial activity against both S. aureus and E. coli signifying them promising for anti-bacterial activity. No relationship was observed between chemotype specificity and anti-bacterial activity, indicating that apart from major components of essential oil, minor components and other factors may be responsible for anti-microbial activities.