Using a daily diary design, the current study assessed within-person associations of work-to-family conflict with negative affect and salivary cortisol. Furthermore, the authors investigated whether supervisor support moderated these associations. Over 8 consecutive days, 131 working parents employed by an information technology company answered telephone interviews about stressors and mood that occurred in the previous 24 hours. On Days 2–4 of the study protocol, they also provided 5 saliva samples throughout the day that were assayed for cortisol. Results indicated a high degree of day-to-day fluctuation in work-to-family conflict, with employed parents having greater negative affect and poorer cortisol regulation on days with higher work-to-family conflict compared to days when they experience lower work-to-family conflict. These associations were buffered, however, when individuals had supervisors who offered support. Discussion centers on the use of dynamic assessments of work-to-family conflict and employee well-being.