The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone-derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell-cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27(Kip1) and p16(Ink4A). These changes are associated with arrest in S-G 2/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors.