OBJECTIVE: Mindfulness practice, a form of meditation, has shown benefit for psychological and physical health. In this study, we investigated the effect of an intensive period of Mindfulness practice on some biological mediators of stress and inflammation during a 3-day residential retreat. METHODS: A total of 95 healthy individuals (aged 18-67) were recruited and randomized to a Mindfulness retreat arm or an active control arm. Before (t0) and after (t1) the intervention, all the participants were assessed for salivary cortisol levels and for a panel of pro- and anti-inflammatory cytokines measured in saliva. Psychometric measures on stress, anxiety and awareness were carried out using PSS, STAI-Y and MAAS questionnaires, respectively. RESULTS: As to the within-group differences, we observed a statistically significant decrease in perceived stress (β = -8.85, p < 0.0001), and anxiety scores (β = -12.39, p < 0.0001), while awareness increased (β = 15.26, p < 0.0001) between t0 to t1 in retreat participants. In the mindfulness intervention group, we also observed a statistically significant reduction in the levels of pro-inflammatory cytokines IL-6 (β = -0.94 p = 0.001) and IL-8 (β = -176.40, p < 0.0001), and an increase in anti-inflammatory IL-10 (β = 0.89 p < 0.0001) levels at the end of the retreat. At t1 we observed a highly significant correlation between cortisol levels and both anxiety (r = 0.56, p < 0.0001) and perceived stress (r = 0.92, p < 0.0001) scores. CONCLUSIONS: Mindfulness retreat participants showed a significant reduction in perceived stress and anxiety levels, as well as an improved balance of some key mediators of inflammatory states. Our data provide evidence that a mindfulness retreat may be effective in improving physical and mental health. Future studies with larger numbers of subjects and follow-up periods may examine mindfulness practice as a non-pharmacological alternative to promote stress reduction and overall health and wellbeing.