It has been hypothesized that incubation begins before laying is completed because it controls bacterial growth on eggshells and infection of eggs. If so, early incubation should be associated with decreased abundance of bacteria on eggshells. There is no detailed information on incubation during daytime or nighttime during laying and its association with bird eggshells' bacterial loads. We used temperature sensors placed in contact with eggs and connected to data-loggers to measure early incubation by females of the Pied Flycatcher, Ficedula hypoleuca, a hole-nesting passerine, beginning with laying of the third egg (day 3). Incubation usually began with laying of the fourth egg (day 4), the modal clutch size in our population being six. We sampled heterotrophic bacteria on eggshells from the whole clutch soon after it was completed. We used colony-forming units counted on trypticase soy agar to estimate bacterial loads. Clutches incubated more intensively during daytime on day 4 and also during nighttime on days 3–5 had fewer bacteria on eggshells than clutches incubated less. The temperature of the air around the eggs seemed not the key explanatory variable for the eggshell's bacterial loads, suggesting that mechanisms other than thermal changes associated with incubation may be more relevant to control of bacterial proliferation. Ours is the first study showing a correlation between increased incubation on the day incubation begins during laying and a reduction in bacteria on the eggshells of wild birds. It supports the hypothesis that early incubation is an antimicrobial defense of the clutch.