This study aimed at assessing the cytotoxicity of Eremomastax speciosa crude extract on NIH-3T3 fibroblast cell lines and reporting the chemical constituents in the extract. The MTT assay on NIH-3T3 cells showed a significantly lower (p < 0.05) inhibition from E. speciosa (IC50 > 30 µg/mL) compared to cyclohexamide (IC50 > 0.8 µg/mL). This result validates the non-toxicity observed with the use of E. speciosa on normal cells at low to moderate doses. Four compounds were isolated and identified from their EIMS as well as 1D and 2D NMR spectroscopic data namely hydroxyandrographolide (1), stigmasterol glucoside (2), (Z)-4-coumaric acid 4-O-β-D-apiofuranosyl-(1’’→2’)-O-β-D-glucopyranoside (3) and 5-methoxy-4,4′-di-O-methyl- secolariciresinol-9′-monoacetate (4). These compounds are isolated from this species for the first time. Thirteen volatile constituents were detected in the extract using gas chromatography mass spectrometry (GC-MS). Besides 6,10,14-trimethy-2-pentadecanone (12.63%), mostly fatty acid esters were detected in high amounts notably ethyl hexadecanoate (16.00%), ethyl-9,12,15-octadecatrienoate (11.51%) and 9,12-octadecadienoic acid ethyl ester (8.05%). This study revealed many unsaturated fatty acid esters in E. speciosa and is noteworthy that ω-3 and ω-6 fatty acid esters were predominant, hence an added nutritional value to this plant.                     KEY WORDS: Eremomastax speciosa, Secondary metabolites, NIH-3T3 cytotoxicity, NMR, GC-MS   Bull. Chem. Soc. Ethiop. 2020, 34(3), 633-640. DOI: https://dx.doi.org/10.4314/bcse.v34i3.18