Genetically superior tea clones United Planters Association of Southern India (UPASI) 3 and UPASI 17 have been identified as prime candidates for chemo-profiling, assessment of antioxidant activity, and evaluation of anticancer properties against the human adenocarcinoma A549 cancer cell line. High-performance liquid chromatography (HPLC) analysis of UPASI 3 and UPASI 17 revealed the presence of caffeine and five catechins, including epicatechin-3-gallate, catechin, epicatechin, epigallocatechin gallate, and epicatechin gallate, with UPASI 17 exhibiting a higher total catechin content than UPASI 3. Comparative assays of antioxidant capacity and tea quality enzymes, such as total polyphenols, polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL), and peroxidase (POD), showed higher levels in UPASI 17 than in UPASI 3. Additionally, transcriptional analysis revealed upregulated expression of key flavonoid biosynthesis genes such as chalcone synthase (CHS), flavonoid 3′,5′-hydroxylase (F3′5′H), anthocyanidin synthase (ANS) dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin 4-reductase (LAR) in UPASI 17 compared with UPASI 3. The anticancer potential of the methanolic extracts of these tea clones against the A549 cancer cell line was evaluated using MTT cytotoxicity assays, along with cell cycle, caspase activation, and mitochondrial membrane potential analyses. Notably, UPASI 17 significantly inhibited cancer cell viability at a concentration of 0.5 µg/ml within 36 h, induced cell cycle arrest in the S phase, and more effectively activated the caspase 3 enzyme to promote apoptosis than UPASI 3. Furthermore, exposure to UPASI 3 and UPASI 17 extracts triggered apoptosis in A549 cells through modulation of the PI3K-Akt-mTOR signalling pathway. The enhanced anticancer activity of UPASI 17 was attributed to its high concentration of phytocompounds and its antioxidant activities. These findings underscore the potential for further molecular-level research and animal studies to develop novel anticancer drugs that target lethal diseases.