Proteasome activity affects cell cycle progression as well as the immune response, and it is largely recognized as an attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudodi/tripeptides bearing at the C-terminal position different α-ketoamide moieties as pharmacophoric units for the interaction with the catalytic threonine residue that sustains the proteolytic action of the proteasome. Among these, we identified the 1-naphthyl derivative 13c as a potent and selective inhibitor of the β5 subunit of the 20S proteasome, exhibiting nanomolar potency in vitro (β5 IC50 = 7 nM, β1 IC50 = 60 μM, β2 IC50 > 100 μM). Furthermore, it significantly inhibited proliferation and induced apoptosis of the human colorectal carcinoma cell line HCT116.