Flash column chromatographic fractionation of tree of heaven (Ailanthus altissima) stem and trunk bark extracts, guided by thin-layer chromatography (TLC)–Bacillus subtilis assay and TLC–heated electrospray high-resolution tandem mass spectrometry (HESI-HRMS/MS), lead to the isolation of six known compounds: (9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (13-HODE, A1), (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9-HODE, A2), hexadecanedioic acid (thapsic acid, A3), 16-hydroxyhexadecanoic acid (juniperic acid, A4), 16-feruloyloxypalmitic acid (alpinagalanate, A5), and canthin-6-one (A6). Their structures were elucidated by HESI-HRMS/MS and one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. This is the first study identifying A1–A5 in A. altissima tree. Except for A5, all isolated compounds exhibited antibacterial activity against B. subtilis in microdilution assays. A6 showed the strongest effect with a minimum inhibitory concentration (MIC) value of 8.3 µg/mL. The antibacterial activity of A3 and A4 is newly described.