The effect of several polyphenols on the 20S proteasomes, both the constitutive and the LMP proteasomes, isolated from bovine tissues, has been investigated. Polyphenolic compounds show many biological activities such as antiviral, antibacterial, antifungal, anti-inflammatory, antimutagenic, and antiallergic activities. However, the molecular mechanism underlying these effects has not been identified. It is well established that polyphenols possess inhibitory activities on several enzymes and among them the 20S proteasome. In the present work, the ChT-L, BrAAP, PGPH, and T-L activities of the isolated constitutive and immuno-proteasomes were assayed in order to get an overall information on the polyphenols binding to the complexes. The effects of the polyphenols on the proteasomal activities were analyzed, taking into account the different subunits composition of the two complexes. Furthermore the same activities were measured on whole extracts from cancer cells exposed to EGCG and gallic acid, evaluating, also, their antioxidant action under oxidative stress. EGCG and gallic acid are able to affect the 20S proteasomes functionality, depending on the complex subunit composition and, in cell extracts, they behave both as antioxidants and proteasome effectors.