The objective of this research was to find the best induction media using 2.4-D (2.4-dichlorophenoxy acetic acid) and NAA (naphthalene acetic acid) for in vitro growth of soybean callus, and to identify the founded antibacterial compounds through callus culture induced by 2,4-D and NAA. This study consisted of three experiments, which were (1) callus culture of soybean using MS basal medium suplemented by 2.4-D and NAA, (2) Extraction of active fraction from callus and test of antibacterial activities using bacterial test of Bacillus substilis dan Escherichia coli, and (3) GC-MS analyses to identify the antibacterial compunds obtained. The research was set up in Paired and Unpaired Test Design with different replications and two treatments of media (MS + 2,4-D 40 mg/l and MS + 2,4-D 5 mg/l + NAA 5 mg/l). The significant differences among treatment means were calculated by the T test (α = 0.05). The results showed that MS + 2,4-D 5 mg/l + NAA 5 mg/l was the better media for inducing callus soybean with immature cotyledone explants. Hexane fraction from callus of MS + 2,4-D 5 mg/l + NAA 5 mg/l exhibited higher antibacterial activity compared to that of MS + 2,4-D 40 mg/l and other fractions. GC-MS analyzes to hexane fraction showed the identified antibacterial compounds, that were : beta-sitosterol, cholesta-3,5-dien-7-one dan phenol (from callus source of MS + 2,4-D 40 mg/l), and oleic acid and cholesta-3,5-dien-7-one (from callus source of MS + 2,4-D 40 mg/l). Antibacterial activities of hexane fraction against Bacillus substilis was more higher than that of Escherichia coli
