Milk thistle (Silybum marianum) is a medicinal plant recognized for its high content of phenolic compounds, notably flavonolignans, flavonoids, and simple phenolic acids, which contribute to its significant antioxidant and hepatoprotective activities. This study aimed to characterize the biochemical composition and to assess the activities of key enzymes—phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO)—involved in phenolic biosynthesis in various plant parts and commercial extracts across different growth stages. Plant samples, including leaves, stems, flowers, seeds, and commercial extracts, were collected monthly over a six-month period, and standard protocols were used for biochemical and enzymatic analyses. Results showed that PPO and PAL activities peaked at the fourth and third month, respectively, then declined, with aerial parts exhibiting higher activity than flowers, seeds, or commercial extracts. Total phenols and flavonoids increased with plant age, while carotenoids, chlorophylls, and β-carotene showed distinct temporal patterns. Notably, commercial extracts demonstrated negligible enzyme activity and generally lower levels of bioactive compounds. Study limitations include limited sampling frequency, absence of environmental data, and a lack of individual phenolic profiling or bioactivity assays. Future research should employ advanced metabolite identification, assess additional enzymes and gene expression, integrate environmental monitoring, and directly evaluate biological activities. These findings provide insight into the dynamic biochemical profile of milk thistle and highlight areas for further investigation to optimize its medicinal use.