Previous studies have demonstrated mindfulness-based programs are effective in reducing workplace stress; however, few have investigated the effectiveness of online mindfulness-based programs. Physiological measures are also underutilised when examining the efficacy of these programs. Therefore, we assessed the efficacy of an online mindfulness-based program on both short-term and more enduring aspects of psychological and physiological measures of stress and ill-health. Participants (N = 22) completed an 8-week online mindfulness-based program. Immediately before and after a mindfulness session, participants completed questionnaires to assess the short-term effects on mood and state anxiety and provided saliva samples to assess salivary alpha amylase (sAA) concentrations. Additionally, pre and post the 8-week intervention, participants completed questionnaires assessing workplace stress, over-commitment, work engagement, dispositional mindfulness and personal resources. Saliva samples were collected to measure the cortisol awakening response, sAA awakening response and secretory immunoglobulin A (sIgA). Our findings demonstrate that online mindfulness-based programs induce short-term improvements in fatigue and anxiety in employees. Additionally, we also report a decrease in over-commitment and increases in optimism and mucosal immunity (sIgA) following the program.