Long term stress exposure is typical for modern societies and might trigger different diseases. This case-control study reveals that persons who had suffered an acute myocardial infarction (AMI) had elevated cortisol concentrations in the month before the acute event. Middle-aged patients admitted to cardiology clinics with acute myocardial infarction (AMI) (n = 174) were compared to 3156 controls from a population-based cohort in southeast Sweden. The median Hair Cortisol Concentrations (HCC) for those who had suffered an AMI was 53.2 pg/mg compared to 22.2 pg/mg for the control group (p < 0.001). In bivariate analysis, higher levels of HCC were strongly (OR = 5.69) and statistically significantly associated with current AMI status. The discrimination of cases with AMI from controls remained statistically significant (OR = 5.04) even after controlling for established cardiovascular risk factors in a multivariate analysis. Middle-aged persons with acute myocardial infarction had significantly elevated cortisol levels during the month before the cardiac event. This was evident for both men and women. The biomarker cortisol concentration was independently and statistically significantly related to AMI. Chronic stress seems to be a new promising risk factor for AMI.