Abstract Sea cucumber Holothuria scabra is a potential export commodity in the fisheries sub-sector. Some are used indirectly in extract form. Selection of the extraction method is carried out by considering its activity. One of the benefits that is often claimed in sea cucumber products is antimicrobial activity. This study aims to determine the antimicrobial activity of sea cucumbers after the extraction process using the reflux method. The ethanol 70% was used as solvent with citric acid as catalyst. Selection of the extraction solvent and catalyst is carried out by considering its safety for human consumption. The catalyst is used to hydrolyze polysaccharides. With the presence of a catalyst combine with various contact times, and temperature we try to assay its microbial activity. We used a fixed amount of solvent and the catalyst concentration was set from 0 to 1% (w/v), contact time 6 to 18 hours, temperature 25 to 55 °C. The experiment design was using factorial analysis at two levels. All the solution was filtered and evaporated to get the dried extract and freeze-dried before it was tested in S. aureus, B. subtilis, and E. coli to see its inhibition zone. The result showed that on SA catalyst and temperature had a significant effect on the inhibition zone. However, on BS and EC, only the catalyst has a significant effect on the inhibition zone. The best-obtained result was estimated using higher catalyst concentration, shorter extraction times, and the lowest temperature.