Background and Objective: Melatonin in breast milk exhibits a 24-hour circadian rhythm, present in nighttime breast milk but nearly undetectable in daytime breast milk. Shift work can disrupt the circadian timing of individuals, evident in changes in melatonin in saliva and urine samples. However, it is unknown whether these changes are also reflected in breast milk from a shift working mother. The aim of this study was to investigate whether maternal circadian rhythm disturbance from shift work impacts the melatonin concentration in breast milk. Materials and Methods: Breast milk and saliva samples were collected from 11 shift working mothers at four timepoints across five consecutive days. This included during their day shift or nonworkdays to act as a control, night shift, subsequent night shifts and postnight shift. Where possible, pre- and postfeed collections were also undertaken. Samples were grouped into four-time intervals: 12–6:30 am, 7–11:30 am, 12–6:30 pm, 7–11:30 pm, and melatonin levels (picogram per milliliter) in the breast milk and saliva samples were analyzed. Results: There was a significant decrease in breast milk melatonin (p = 0.026) at the 12–6:30 am time interval on subsequent night shifts, compared with control days. However, there was no overall time and shift type interaction effect (p = 0.70). In addition, no observed difference in melatonin levels was found in saliva samples, or when comparing pre- and postfeed breast milk. Breast milk melatonin however was found to be significantly higher compared with saliva (p > 0.001), at all but one time interval. Conclusion: The findings suggest that there is a potential effect of maternal circadian rhythm disruption from shift work on breast milk melatonin levels. This is an important step in exploring the role of maternal circadian timing and the effect on breast milk composition. Expansion of this research and exploration of other circadian rhythm misalignment sleep disorders on breast milk is highly recommended.