OBJECTIVE: Heart rate variability (HRV) and mindfulness have been described as correlates of self-regulation and well-being. The goal of the present study was to investigate their interactions from a within-person perspective in the context of work. METHODS: Applying an ambulatory assessment approach, we studied 89 healthcare professionals across two to four work shifts. Self-reports of momentary job demands, mindfulness, and well-being (as indicated by emotional exhaustion, relaxation, and contentment) were provided three to four times a day via smartphone questionnaires. Electrocardiogram and activity sensors continuously recorded data from the beginning to the end of the shifts. Multilevel models based on 937 measurements were built for emotional exhaustion, relaxation, and contentment. RESULTS: After controlling for covariates, including bodily movement, shift, and job demands, short-term HRV was marginally significantly related to decreased emotional exhaustion and significantly related to increased relaxation. State mindfulness was significantly related to decreased emotional exhaustion, and increased relaxation and contentment. Furthermore, HRV and mindfulness significantly interacted such that emotional exhaustion was lowest and relaxation was highest when both HRV and mindfulness were high. CONCLUSIONS: Together, the findings provide insights into the use of HRV and mindfulness as indexes of psychophysiological regulatory resources that seemingly intensify their respective beneficial effects on the daily well-being of employees.