We conducted studies with high‐molecular‐weight (HMW) and low‐molecular‐weight (LMW), hydrophobic, and hydrophilic dissolved organic matter (DOM) from an oligotrophic alpine river, the Tagliamento River (Italy), to assess the effect of light on DOM utilization by riverine bacterioplankton. Immediately after the exposure of the DOM fractions to simulated or natural sunlight, short‐term (1 h) bacterial utilization of all DOM fractions decreased by up to 80%, compared with the uptake of the corresponding nonirradiated fractions. The addition of scavengers suggests that reactive oxygen species caused this bacterial growth inhibition. After the long‐term growth of bacteria on irradiated DOM, uptake was unchanged for HMW DOM, considerably lower for LMW and hydrophilic DOM, and much higher for hydrophobic DOM, compared with the nonirradiated fractions. These results suggest that the phototransformation of HMW, LMW, hydrophobic, and hydrophilic DOM results in contrasting effects on their bioavailability. Size‐exclusion chromatography showed that bacteria preferably used the larger molecular sizes of all nonirradiated fractions. Light induced no significant shifts in the apparent molecular weight distribution of the four DOM fractions. However, the highly bioavailable LMW DOM (especially the portion ~5 kDa) was no longer taken up after irradiation. We conclude that light overall leads to a strong decrease in microbial DOM transformation during hydrological transport in the Tagliamento River.