Eleven Thai medicinal plants extracted with 95% ethanol were tested for: antibacterial activity against Staphylococcus aureus ATCC 25923, Streptococcus pyogenes A 6/49, Haemophilus influenzaeJ Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC 10031. Most of the extracts showed good activity against gram positive bacteria and only a few of them showed activity against gram negative bacteria. Seven plant extracts were selected and extracted with petroleum ether, chloroform and ethanol. The residues were tested for antibacterial activity. Eleutherine palmifolia (L.) Merr. bulb with petroleum ether extraction provided the antibacterial activity with the lowest concentration. Eleutherine palmifolia (L.) Merr. with petroleum ether extraction was isolated by column chromatography technique for antibacterial substance and was identified by spectroscopy. The new compound which had never been found in plant was yellow needle crystal. Its structure was 1,4-naphthoquinone derivative. The minimal inhibitory concentration of the pure compound against S. aureus and S. pyogenes were 40 μg/ml and 60 μg/ml. However, the MIC90 of this compound against S. aureus and S. pyogenes were 29.2 μg/ml and 39.0 μg/ml, respectively.