Studies have documented various effects of circadian rhythm and daytime variations on the cardiovascular and immune system as well as wound healing. From June to December 2016, n = 367 cardiac surgery patients were enrolled. Microbiological swabs from the mediastinum and subcutaneous wound were taken before sternal closure. Patients were assigned to groups based on operation start: morning (n = 219) or afternoon (n = 135). Bacterial contamination and wound infections were studied in relation to circadian rhythm and daytime variation. We did not observe any difference in mortality (morning: 3.7%, afternoon: 3.0%, p > 0.99) and major adverse events (morning: 8.2%, afternoon: 5.9%, p = 0.53). In 27.7% of the morning group, at least one positive intraoperative swab was observed, similar to the afternoon group (25.6%, p = 0.71). The incidence of positive presternal swabs was 15.6% in the morning compared to 9.1% in the afternoon (p = 0.18). About 90% of the germs detected were part of the natural skin flora (e.g., Cutibacterium acnes and Staphylococcus epidermidis). The incidence of sternal wound infections was 7.3% (morning) and 3.0% (afternoon) (p = 0.18). We did not find differences in the incidence of intraoperative bacterial sternal contamination, nor postoperative infections, between patients who underwent cardiac surgery in the morning or afternoon.