Introduction: This study aimed to identify the chemical compounds and examine the in vitro anti-inflammatory activity of Geniotrigona thoracica propolis extract in inhibiting nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages. Methods: Total phenolic and flavonoid content was determined using spectrophotometric methods. Prior to anti-inflammatory studies, cytotoxicity test was performed to determine the safest propolis concentration for RAW 264.7 cells. Anti-inflammatory activity tests including NO production assay (Griess assay), iNOS production and TNF-α production using Enzyme linked Immunosorbent Assay (ELISA) kit. The chemical components of the propolis extract were identified by Liquid chromatography tandem mass spectrometry (LC-MS/MS) technique. Results: The TPC and TFC were 374.20 ± 0.51 mg GAE/g extract and 20.66 ± 4.08mg QE/g extract, respectively. Propolis extract at concentrations of 12.5, 25, and 50 μg/mL was non-toxic, making it suitable for anti-inflammatory testing. The anti-inflammatory test results showed inhibition of TNF-α at 12.5 μg/mL EEP, which was 11.06%, inhibition of NO at 50 μg/mL, which was 22%, and inhibition of iNOS at 12.5 μg/mL, which was 41%. LC-MS/MS analysis revealed that propolis extract includes anti-inflammatory bioactive substances such as β-mangostin, garcinone D, garcinone E, DL-stachydrine, betaine, salvinorin-A, mangostin, and 18-β-glycyrrhetinic acid. Conclusion: Propolis extract from Geniotrigona thoracica, Pematang Siantar area, North Sumatra, Indonesia, has anti-inflammatory bioactive compounds in the results of the LCMS/MS method, and the results of the anti-inflammatory activity on TNF-α, NO, and iNOS indicate the presence of anti-inflammatory activity in the extract. © 2025 Universiti Putra Malaysia Press. All rights reserved.