Isolation and determination of antibacterial compounds from plants are essential to obtain a new antibacterial as a substitute for conventional resistant antibiotics. This study aims to isolate and identify a new robustaflavone as antibacterial activity from Garcinia latissima Miq. leave. In this study, the isolation process was carried out using column chromatography followed by preparative thin layer chromatography (TLC) based on the TLC profile. The fraction D was tested for anti-bacterial Bacillus subtilis using the TLC bioautography method. The isolates obtained were then identified using 1H-NMR, 13C-NMR, distortionless enhancement by polarization transfer, heteronuclear single quantum coherence, and heteronuclear multiple bond coherence. The Activity assay of the isolate was performed using the microdilution method. A pure compound obtained the result of the separation process with eluent n-hexane: Ethyl acetate (3:2) with Rf 0.6. This spot follows the spot in the contact bioautographic result of fraction D, the spot with Rf 0.6 gives an inhibition zone. After identifying and purifying the isolate were known as Robustaflavone, this compound has activity against B. subtilis with a (minimum inhibitory concentration) value of 2500 ppm. Robustaflavone successfully isolated and identified from G. latissima leave and its antibacterial activity.