The first phytochemical investigation of the leaves of Fissistigma fulgens (Hook.f. & Thomson) Merr. led to the isolation and identification of five previously undescribed dihydrochalcone derivatives, including two dimeric dihydrochalcones, fissisfulgenones A and B (1 and 2), and three monomeric dihydrochalcones, fissisfulgenones C-E (3-5), along with three known compounds. Their structures were elucidated by spectroscopic analysis. The dimeric structure of fissisfulgenone A (1) was confirmed by single-crystal X-ray diffraction analysis using Cu-Kα radiation. The pure enantiomers of the scalemic mixtures of 1-3 were successfully resolved using chiral-phase HPLC, and their absolute configurations were determined by comparing experimental and calculated ECD spectra. Fissisfulgenones A (1), C (3), and D (4) were evaluated for biological activities, including cytotoxicity against human breast adenocarcinoma (MDA-MB231), human lung carcinoma (A549), human hepatocellular carcinoma (Huh7), and human colorectal adenocarcinoma (SW480 and HT29), antiviral activity against dengue virus (DENV), and nitric oxide production inhibitory activity in LPS-stimulated RAW 264.7 macrophages. Among them, fissisfulgenone C (3) exhibited the highest cytotoxicity against SW480 and HT29 cells with IC50 values of 40.6 and 68.0 μM, respectively, whereas fissisfulgenone D (4) showed cytotoxicity against MDA-MB231, A549, and Huh7 cell lines with IC50 values of 48.7, 38.3, and 21.2 μM, respectively. Notably, fissisfulgenone A (1) significantly reduced virus production in DENV-infected Huh7 cells at a sub-toxic dose of 50.0 μM. Regarding anti-inflammatory activity, fissisfulgenone C (3) exhibited strong nitric oxide inhibition, markedly reducing LPS-induced levels with an IC50 value of 5.27 μM and preserving normal macrophage morphology. Fissisfulgenone A (1) also showed similar effects at lower, non-toxic concentrations.