Chalcones are aromatic ketones, known to exhibit anti-microbial, anti-inflammatory and anti-cancer activities. The aim of this study was to investigate the anti-inflammatory and anti-cancer activity of 4'-hydroxychalcone. Here, we report that 4'-hydroxychalcone inhibits TNFα-induced NF-κB pathway activation in a dose-dependent manner. To investigate the underlying molecular mechanisms we demonstrate that 4'-hydroxychalcone inhibits proteasome activity in a dose-dependent manner but has no effect on IKK activity. Results show that 4'-hydroxychalcone inhibits TNFα-dependent degradation of IκBα and subsequently prevents p50/p65 nuclear translocation leading to 4'-hydroxychalcone-inhibited expression of NF-κB target genes. Most importantly, inhibition of NF-κB activation by 4'-hydroxychalcone is not leukemia cell-type specific and has no significant effect on non-transformed cell viability, thus highlighting the compound's potential in both prevention and treatment.