Achillea biebersteinii is a perennial medicinal plant traditionally used in various countries for the treatment of several diseases and is rich in phenolic compounds with reported anticancer potential. However, its molecular mechanisms against ependymal tumors remain poorly understood. This study aimed to investigate the anticarcinogenic effects of Achillea biebersteinii aqueous extract on DKFZ-EP1NS human ependymal tumor cells, with a focus on transient receptor potential canonical (TRPC6) channels and the nuclear factor kappa B (NF-κB) signaling pathway. The phenolic composition of the extract was determined by high-performance liquid chromatography (HPLC), and its cytotoxicity was assessed using the MTT assay. Apoptotic activity and cell cycle distribution were evaluated by flow cytometry, while TRP channel currents were recorded via the whole-cell patch-clamp technique. TRPC6 and NF-κB gene expression levels were quantified by real-time PCR. The extract demonstrated concentration-dependent cytotoxicity, with an IC50 value of 0.241 mg/mL (p < 0.05). It induced early apoptosis in over 70% of cells (p < 0.05), caused significant G0/G1 phase cell cycle arrest (p < 0.05), and markedly inhibited TRP channel conductance and current amplitudes (p < 0.05). Gene expression analysis showed significant downregulation of TRPC6 and NF-κB transcripts, with fold changes ranging from 5.6- to 23.7-fold for NF-κB and 8.4- to 10.9-fold for TRPC6 (p < 0.05). These results suggest that Achillea biebersteinii aqueous extract inhibits TRP channel activity and suppresses NF-κB signaling, thereby reducing cell proliferation through apoptosis induction and cell cycle arrest, providing clues to its possible anticancer mechanism and supporting its potential as a phytotherapeutic candidate in ependymoma treatment.