Background Inverse associations of the Mediterranean diet (MedDiet) and physical activity with cardiovascular disease have been previously reported. We investigated the individual and combined contributions of both to this inverse association in a Mediterranean cohort. Design We used data from 19,536 participants from a prospective cohort of Spanish university graduates, the 'Seguimiento Universidad de Navarra' (SUN) cohort, followed up between December 1999 and December 2016. Methods Adherence to the MedDiet was obtained from a 136-item validated food-frequency questionnaire and categorized in tertiles using four previously reported dietary scores. A validated questionnaire assessed the physical activity levels according to volume, intensity and frequency. Results Participants were followed up during a median time of 10.4 years. Compared with the lowest category of adherence to the MedDiet (≤3 in the Mediterranean Diet Score), higher adherence (6-9 points) was strongly inversely associated with cardiovascular disease (multivariable adjusted hazard ratio = 0.33; 95% confidence interval (CI) 0.20-0.55). Also, engaging in an active lifestyle (6-8 points in an eight-item score) compared with low activity (<2 points) was associated with a reduced risk of incident cardiovascular disease (hazard ratio = 0.43; 95% CI 0.20-0.90). Greater adherence to the MedDiet and engaging in high levels of active lifestyle showed a 75% relatively reduced risk of cardiovascular disease (hazard ratio = 0.25; 95% CI 0.13-0.48). Conclusions The combined effect of adherence to the MedDiet and adopting an active lifestyle showed a synergistic inverse association with cardiovascular disease risk.