High work stress has been consistently associated with disturbed autonomic balance, specifically, lowered vagal cardiac control and increased sympathetic activity, which may lead to increased cardiovascular risk. Stress management procedures have been proposed to reduce autonomic dysfunctions related to work stress in different categories of workers exposed to heightened work demands, while a limited number of studies addressed this issue in managers. The present study was aimed at evaluating the effectiveness of a respiratory sinus arrhythmia (RSA) biofeedback (BF) intervention on psychological and physiological outcomes, in managers with high-level work responsibilities. Thirty-one managers leading outstanding private or public companies were randomly assigned to either a RSA-BF training (RSA-BF; N = 16) or a control group (N = 15). The RSA-BF training consisted of five weekly 45 min sessions, designed to increase RSA, whereas controls had to provide a daily stress diary once a week. After the training, managers in both groups reported reduced heart rate at rest, lower anxiety levels and improvement in health-related quality of life. More importantly, managers in the RSA-BF group showed increased vagal control (as indexed by increased RSA), decreased sympathetic arousal (as indexed by reduced skin conductance and systolic blood pressure) and lower emotional interferences, compared to managers in the control group. Results from this study showed that RSA-BF training was effective in improving cardiac autonomic balance at rest. Moreover, findings from this study underline the effectiveness of biofeedback in reducing psychophysiological negative outcomes associated with stress in managers.