Summary The anthroposophic lifestyle implies environmental conditions for the infant aimed at reducing negative stress stimulation and is also related to a lower prevalence of allergic diseases in children. One aim of this prospective birth cohort study was to assess stress levels in infants with an anthroposophic lifestyle. A total of 330 infants from families with anthroposophic or more conventional lifestyles were followed from pregnancy of their mothers until the age of 6 months. Information on lifestyle factors was obtained from questionnaires. Salivary samples from 210 6-month olds and their parents were collected on three occasions during 1 day for analysis of cortisol. Infants from families with an anthroposophic lifestyle had significantly lower cortisol levels on all three sampling occasions compared to other infants. In the morning, the geometric means of salivary cortisol concentration (with 95% confidence limits) were 8.8 nmol/l (6.7–11.5), 11.3 nmol/l (9.3–13.7) and 14.9 nmol/l (11.3–19.6) in infants classified as anthroposophic, partly anthroposophic and non-anthroposophic, respectively ( p = 0.018). On the other hand, there was no difference in cortisol levels between the parents in the different groups. Several lifestyle factors differed significantly between the groups, but none of them independently explained the difference in cortisol levels. However, living on a farm during pregnancy was significantly associated with low saliva cortisol level in the infant. It can be concluded that low salivary cortisol levels in infants from anthroposophic families may be related to an environment with a lower degree of exposure to stress, which could influence the development of allergic diseases.