Although compelling evidence exists on the ability of aspirin to treat colorectal cancer (CRC), and numerous theories and targets have been proposed, a consensus has not been reached regarding its mechanism of action. In this regard, a relatively unexplored area is the role played by aspirin metabolites 2,3‑dihydroxybenzoic acid (2,3‑DHBA) and 2,5‑dihydroxybenzoic acid (2,5‑DHBA) in its chemopreventive actions. In a previous study, we demonstrated that 2,3‑DHBA and 2,5‑DHBA inhibited CDK1 enzyme activity in vitro. The aim of the present study was to understand the effect of these metabolites on the enzyme activity of all CDKs involved in cell cycle regulation (CDKs 1, 2, 4 and 6) as well as their effect on clonal formation in three different cancer cell lines. Additionally, in silico studies were performed to determine the potential sites of interactions of 2,3‑DHBA and 2,5‑DHBA with CDKs. We demonstrated that 2,3‑DHBA and 2,5‑DHBA inhibits CDK‑1 enzyme activity beginning at 500 µM, while CDK2 and CDK4 activity was inhibited only at higher concentrations (>750 µM). 2,3‑DHBA inhibited CDK6 enzyme activity from 250 µM, while 2,5‑DHBA inhibited its activity >750 µM. Colony formation assays showed that 2,5‑DHBA was highly effective in inhibiting clonal formation in HCT‑116 and HT‑29 CRC cell lines (250‑500 µM), and in the MDA‑MB‑231 breast cancer cell line (~100 µM). In contrast 2,3‑DHBA was effective only in MDA‑MB‑231 cells (~500 µM). Both aspirin and salicylic acid failed to inhibit all four CDKs and colony formation. Based on the present results, it is suggested that 2,3‑DHBA and 2,5‑DHBA may contribute to the chemopreventive properties of aspirin, possibly through the inhibition of CDKs. The present data and the proposed mechanisms should open new areas for future investigations.