Sea cucumbers produce various bioactive compounds especially secondary metabolites including Saponin. The aim of this study was to investigate antibacterial activity of the Saponin extracted from the Persian Gulf sea cucumber (Stichopus hermanni) in some gram positive and negative bacteria. Extraction was first done by ethanol 70% and then Saponins were separated through column chromatography with silica gel. Extracted Saponins were identified through thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were investigated via tubular dilution. Two groups of steroidal and glycosides Saponins with R_f 0.9 and 0.05 were respectively identified in fractions extracted by TLC. HPTLC results for a sample of extracted Saponin confirmed the existence of Saponin in sea cucumber regarding the R_f. The gram negative bacterium Pseudomonas aeroginosa was the most resistant one to steroidal and glycosides- steroidal Saponins with MIC of 500 and 400 micg/ml, respectively. Steroidal and glycosides- steroidal Saponins showed no bactericidal effect in gram negative bacterium Pseudomonas aeroginosa. Among the gram positive bacteria, the highest MBC of 500 micg/ml belonged to glycosides- steroidal Saponin for Staphylococcus aureus. The higher MIC in gram negative bacteria approves the lower antibacterial property of the steroidal and glycosides Saponin extracted from sea cucumber Stichopus hermanni in these bacteria compared to the gram positive ones.