Teucrium polium is a medicinal plant widely used in traditional medicine and is known for its diverse pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. This study investigates the anticancer potential of T. polium extracts incorporated into silver nanoparticles (AgNPs). Following extraction, the total phenolic content (TPC) and total flavonoid content (TFC) of the extracts were quantified, and their chemical profiles were analyzed using UVVis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), liquid chromatographymass spectrometry (LC-MS), and high-performance liquid chromatography (HPLC). Among the tested solvents, the ethanolic extract exhibited the highest TPC (140.2 ± 2.4 mg GAE/g), followed by the aqueous extract (107.2 ± 1.8 mg GAE/g), while the hexane extract contained negligible phenolic content. Consequently, only the ethanolic and aqueous extracts were utilized for AgNP synthesis. The synthesis of AgNPs was optimized by varying the extract concentration, pH, and incubation time. The most stable and optimal nanoparticles were obtained using a T. polium extract concentration of 10 mg/mL, a pH of 10.5, and an incubation period of 4 hours. These optimized AgNPs were subsequently evaluated for anticancer activity. Cytotoxicity assays demonstrated that ethanolic AgNPs exhibited higher toxicity toward Caco-2 colorectal cancer cells compared to CCD-841 normal colon cells. At the highest concentration tested (10 μg/mL), Caco-2 cell viability was significantly reduced to 11.15 ± 0.69%, whereas CCD-841 cells retained a higher viability of 58.54 ± 2.98%. At the lowest concentration (0.3125 μg/mL), ethanolic AgNPs had minimal cytotoxic effects, with Caco-2 cell viability at 59.79 ± 1.62% and CCD-841 cell viability at 92.11 ± 0.95%. These findings suggest that CCD-841 cells exhibit lower sensitivity to AgNP exposure at sub-lethal concentrations compared to Caco-2 cells, highlighting the potential selectivity of T. poliumbased AgNPs for cancer therapy.