Context: Preparations from Artocarpus heterophyllus Lam. (Moraceae) heartwoods are used in the traditional folk medicine for the treatment of inflammation, malarial fever, and to prevent bacterial and fungal infections. Objective: The objective of this study was to isolate pure antibacterial compounds from A. heterophyllus heartwoods. Materials and methods: The dried and powdered A. heterophyllus heartwoods were successively extracted with the following solvents: hexane, ethyl acetate, and methanol. Each of the extracts was screened for their antibacterial activities using a disc diffusion method (10 mg/disc). Their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined using a broth microdilution method. The extract that showed the strongest antibacterial activities was fractionated to isolate the active compounds by an antibacterial assay-guided isolation process. Results and discussion: The ethyl acetate extract exhibited the strongest antibacterial activities against Streptococcus mutans, S. pyogenes, and Bacillus subtilis with MIC values of 78, 39, and 9.8 mg/mL, respectively. Based on an antibacterial assay-guided isolation, four antibacterial compounds: cycloartocarpin (1), artocarpin (2), artocarpanone (3), and cyanomaclurin (4) were purified. Among these isolated compounds, artocarpin exhibited the strongest antibacterial activity against Gram-positive bacteria, including S. mutans, S. pyogenes, B. subtilis, Staphylococcus aureus, and S. epidermidis with MICs of 4. 4, 4.4, 17.8, 8.9, and 8.9 mM, respectively, and MBCs of 8.9, 8.9, 17.8, 8.9, and 8.9 mM, respectively, while artocarpanone showed the strongest activity against Escherichia coli, a Gram-negative bacteria with MIC and MBC values of 12.9 and 25.8 mM, respectively. Only artocarpin showed inhibitory activity against Pseudomonas aeruginosa with an MIC of 286.4 mM.