Black sea cucumber (Holothuria atra) is known to have antibacterial substances. The purpose of this study was to investigate the antibacterial effects of Holothuria atra on the growth of Edwarsiella tarda in vitro. Qualitative tests of ethanol extract of Holuthuria atra showed positive tests for alkaloids, saponins and terpenoids, and negative for tannins and flavonoids. Ethanol extract of Holuthuria atra was separated by column chromatography with chloroform:methanol:Aqua Dest (3:5:1)(v/v) as mobile phase, which obtained four extracts, orange (Rf 0.51), light orange (Rf 0.57), yellow (Rf 0.23) and+ light yellow (Rf 0.14). These extracts were compared to tetracycline for antibacterial activity toward Edwardsiella tarda. Orange extract of Holothuria atra at 10 ppm and tetracycline at 5 ppm had the same antibacterial activity. Maximum absorption with UV-Vis of this extract was at 233 nm. Tests with FTIR showed O-H (3300 nm), aliphatic C-H (3010 nm), aromatic C-H (2854 nm and 977 nm), alkene (2923 nm and 2560 nm), C=O (1737 nm), unsaturated alkene with aromatic ring (1625 nm), sulfate (1402 nm, 1201 nm and 1317 nm) and C-O (1263 nm), which strengthens the estimation that the antibacterial ingredient of Holothuria atra is a triterpenoid. LCMS of the orange fraction had two peaks with retention times of 1.9 and 4.1. The highest peak of the orange fraction was at base peak 271.64 m/z and molecular weight 764 g/mol, which suggests a triterpenoid.

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