Wolbachia are ubiquitous bacterial endosymbionts of arthropods and affect host gene expression. Although Wolbachia infections were suggested to modulate sleep in flies, their influence on the circadian clock remained obscure. Here, we screened bacterial symbionts in a laboratory Drosophila melanogaster colony, and observed widespread infections of wMel strain Wolbachia. We established a Wolbachia-free strain from a clock gene reporter strain, period-luciferase (per-luc). Temperature (19–29 °C)-compensated free-running periods were detected regardless of infections which may reflect the lack of wMel infections in central circadian pacemaker neurons. However, locomotor activity levels during the night or subjective night were significantly amplified in uninfected flies. Moreover, the behavioral phenotype of F1 offspring of an uninfected female and infected male resembled that of uninfected flies. This trait is consistent with maternal transmission of Wolbachia infection. Interestingly, per-luc activities in headless bodies, as an index of peripheral circadian oscillators, were severely damped in uninfected flies. Additionally, circadian amplitudes of PER immunoreactivities in Malpighian tubules were reduced in uninfected flies. These results demonstrate that Wolbachia boost fly peripheral clock oscillations and diurnal behavioral patterns. Genetic mechanisms underlying behavioral rhythms have been widely analyzed using mutant flies whereas screening of Wolbachia will be necessary for future studies.