Two diel cycle studies were conducted to determine the effect of day—night light changes on winter bacterial activity in the coastal Mediterranean (Blanes Bay Microbial Observatory). Bacterial abundances, bacterial heterotrophic activity, and flagellate grazing counts were determined at 4-h intervals during two 3-d periods separated by 2 d. Twice a day, the single-cell activity of major bacterial groups was further analyzed by applying microautoradiography combined with catalyzed reporter deposition—fluorescence in situ hybridization. During the first cycle, all the measured parameters (including the number of active cells in most groups) varied synchronously, with higher values at night and lower during the day. An episode of strong winds between the two studied periods disrupted this periodicity at the onset of the second cycle. The bulk incorporation of 3 H-leucine recovered the diel pattern after 2 d, mostly driven by the activity of Gammaproteobacteria. Among the possibilities considered, the observed nighttime increases in the grazing activity of heterotrophic nanoflagellates appeared to partially drive the activity of all bacterial taxa through potential periodic release of dissolved organic matter.