The chemopreventive effects of β-glucan-rich cereals such as oat and barley (beta®barley) have been examined previously, but studies comparing fermentation characteristics and chemopreventive effects of oat and barley of different processing stages are rare. Therefore, the present study aims at investigating the fermentation end points (pH values, concentrations of short-chain fatty acids (SCFA) and ammonia) in fermentation supernatants (FS) obtained from differently processed oat and barley samples (kernels, thick and thin flakes). Chemopreventive effects of FS, such as growth inhibition, apoptosis, and induction of cell cycle- and redox-relevant genes (p21, SOD2), were analysed in LT97 colon adenoma cells. After fermentation, pH values were reduced (∆ pH − 1.3, on average) and SCFA concentrations were increased (∆ + 59 mmol/L, on average) with a shift towards butyrate formation in FS obtained from oat and barley samples compared to the fermentation negative control (FS blank). Ammonia was reduced more effectively in FS obtained from barley (∆ − 4.6 mmol/L, on average) than from oat samples (∆ − 1.0 mmol/L, on average). Treatment of LT97 cells with FS resulted in a time- and dose-dependent reduction of cell number, an increase in caspase-3 activity (up to 9.0-fold after 24 h, on average) and an induction of p21 (2.1-fold, on average) and SOD2 (2.3-fold, on average) mRNA expression, while no genotoxic effects were observed. In general, the results indicate no concrete effect of the type of cereal or processing stage on fermentation and chemopreventive effects of oat and barley.