Background: Work and family are two fundamental aspects of adult life and the conflict between them is considered a source of stress. Work-family conflict (WFC) has been found to be negatively associated with a number of health conditions including psychological distress in a number of studies. Conflict can occur in the direction of work to family interference or from family to work interference, but many studies have not examined both directions of interference in the same population. Furthermore, the association between biological markers of stress and work-family conflict has not been examined. This is key in improving our understanding of the biological pathways involved in the experience of WFC and the manifestation of health conditions. Methodology: Data from the Whitehall II study of British civil servants were used to explore the associations between work-family conflict, biological markers of stress (diurnal cortisol profile, C-reactive protein (CRP) and interleukin-6 (IL-6)) and psychological distress (GHQ). Work-family conflict was assessed using a series of 8 questions, four measure work to family interference and four measure family to work interference. The data used were from phases 3 (1991-1994), 5 (1997-1999) and 7 (2001-2004) of the study. Multiple linear regression, growth curve modelling and cross-lagged models were used through the Stata programme. Results: Analysis suggested that there were cross-sectional associations between work-family conflict and psychological distress. There was no association between work-family conflict and cortisol and there were mixed associations with inflammatory markers. The longitudinal analysis suggested that work-family conflict is associated with psychological distress but there was no association with inflammatory markers. Lastly, findings from the bi-directional analysis indicated that work-family conflict predicts psychological distress and vice versa to a lesser extent. Conclusions: In conclusion, the findings suggest that experiencing work-family conflict is associated with psychological distress. However, the association between WFC and inflammatory markers is inconsistent, and they do not mediate the associations of WFC and psychological distress. These findings could encourage employers to implement workplace policies which minimise conflict between family life, in order to reduce adverse mental health related outcomes. Policies such as flexible working could reduce the WFC that individual's experience.