Abstract: This study investigated the secondary metabolites of the symbiotic fungus Penicillium citrinum MDH-10 from Digitalis purpurea L. First, liquid chromatography-mass spectrometry was employed for auxiliary analysis, after which the ethyl acetate extract of the fungal fermentation broth was separated and purified by silica gel column chromatography, gel column chromatography, and semi-preparative liquid chromatography. Ten compounds were successfully identified through spectroscopic methods (NMR, MS, and IR) and quantum chemical calculations, including one new compound, 4-desacetoxymethyl-11-nor-moniliphenone (4), as well as nine known compounds: monodictyphenone (1), 2,2′,6′-trihydroxy-4-methyl-6-methoxy-acyl-diphenylmethanone (2), benzoic acid,2-(2,6-dihydroxybenzoyl)3-hydroxy-5-(hydroxymethyl)-methyl, methyl ester (3), penicillone C (5), 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (6), 2,5-dimethyl-7-hydroxychromone (7), 12R-aloesol (8), cassiachromone (9), and 7-hydroxy-5-(4-hydroxy-2-methyl-4H-chromen-4-one) (10). Additionally, all compounds were tested for cytotoxic activity against five tumor cell lines (SY5Y, HeLa, HepG2, MIAPaCa-2, and PANC-1). The results indicated that compound 6 exhibited inhibitory activity against the SY5Y neuroblastoma cell line, with an IC₅₀ value of (37±1.7)μmol/L. This study identified structurally novel metabolites from Penicillium citrinum MDH-10, enriching the resource pool of microbial-derived natural products and providing theoretical support for their potential applications.