This study aims to explore the toxic components and mechanism of Dryopteridis Crassirhizomatis Rhizoma based on network pharmacology with experimental validation. An oral acute toxicity test in mice was employed to evaluate the toxicity of different extracted fractions of Dryopteridis Crassirhizomatis Rhizoma, including: 95% ethanol cold soaked percolation extract (EC), 95% ethanol heated reflux extract (EH), water decoction extract (WD). Ultra-high performance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-ESI-MS/MS) was used to analyze the blood-absorbed components after oral administration of Dryopteridis Crassirhizomatis Rhizoma extracts in mice, investigating their major metabolic processes in vivo. Network pharmacology was applied to predict and screen the key toxic components, potential targets, and signaling pathways responsible for the toxicity of Dryopteridis Crassirhizomatis Rhizoma. Molecular docking and zebrafish acute toxicity tests were conducted for further validation. The experimental results demonstrated that the median lethal dose (LD₅₀) of the EC group in mice was 4.30 g/kg, indicating toxicity, whereas the EH group and WD group exhibited LD₅₀ values >20.00 g/kg, classifying them as non-toxic. A total of 16 prototype compounds and metabolites were detected in the serum, most of which were phloroglucinol derivatives. Network pharmacology analysis identified the key toxic components of Dryopteridis Crassirhizomatis Rhizoma as dryocrassin B, 1-methyl-3-butyrylphloroglucinol, flavaspidic acid AB. The core targets associated with toxicity included epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), B-cell lymphoma-2 gene (BCL-2). GO enrichment analysis revealed that the primary biological processes involved were protein phosphorylation, response to exogenous stimuli, collagen catabolic process, rhythmic processes. KEGG pathway analysis indicated significant associations with endocrine resistance pathway, pathways in cancer, lipid and atherosclerosis signaling pathways. Molecular docking further confirmed strong binding affinities between key toxic components (e.g., flavaspidic acid AB, flavaspidic acid PB) and core targets (e.g., EGFR, matrix metallopeptidase 9 (MMP9)). Zebrafish acute toxicity testing showed that flavaspidic acid AB had a median lethal concentration (LC₅₀) of 2.93 μg/mL, classifying it as highly toxic. This study preliminarily elucidated the toxic material basis and potential mechanisms of Dryopteridis Crassirhizomatis Rhizoma, providing a scientific reference for its clinical safety.