Background: High physiological 16α-[18F]-fluoro-17β-estradiol ([18F]-FES) uptake in the abdomen is a limitation of this positron emission tomography (PET) tracer. Therefore, we investigated the effect of food intake prior to PET acquisition on abdominal background activity in [18F]-FES-PET scans. Methods: Breast cancer patients referred for [18F]-FES-PET were included. Three groups were designed: (1) patients who consumed a chocolate bar (fatty meal) between tracer injection and imaging (n = 20), (2) patients who fasted before imaging (n = 20), and (3) patients without diet restrictions (control group, n = 20). We compared the physiological [18F]-FES uptake, expressed as mean standardized uptake value (SUVmean), in the abdomen between groups. Results: A significant difference in [18F]-FES uptake in the gall bladder and stomach lumen was observed between groups, with the lowest values for the chocolate group and highest for the fasting group (p = 0.015 and p = 0.011, respectively). Post hoc analysis showed significant differences in the SUVmean of these organs between the chocolate and fasting groups, but not between the chocolate and control groups. Conclusion: This exploratory study showed that, compared to fasting, eating chocolate decreases physiological gall bladder and stomach [18F]-FES uptake; further reduction through a normal diet was not seen. A prospective study is warranted to confirm this finding.