Abstract: Ascidians are rich source of bioactive agent which could be used for novel antimicrobial drugs. Ascidians are belongs to phylum chordata and class ascidiacea. In the present study a compound ascidia Aplidium multiplicatum, collected from Vizhinjam, south west coast of India was assayed for their antibacterial activity against six human bacterial pathogens. The antibacterial activity of crude extract of ascidians showed inhibitory activity against all six species. The crude methanol extract was more active exhibiting a broad spectrum antibacterial activity than the crude ethanol and acetone extract against each of the bacterial species tested. In antibacterial activity the gram negative bacteria Pseudomonas aeruginosa showed most sensitive against 12.0533± 0.010116 mm A.multiplicatum in crude methanol extract. And the minimum zone of 1.06± 0.121244 mm in K.pneumoniae in ethanol extract. The corresponding zones of ethanol extract produced a maximum zone of 10.3533± 0.7274 mm in Proteus mirabilis and acetone extract produced a maximum zone of 10.143± 0.1266 mm against Pseudomonas aureus. These results indicated that the ascidian A.multiplicatum is found to have remarkable antimicrobial activities against isolated microbes. Further, studies will fulfill for purification and structural elucidation of antimicrobial drugs.