In the ‘One Health’ concept, animal health is linked to human health, through the matrix of food (including the food-processing environment). Application of molecular methods for pathogen analysis of food is, therefore, important in the ‘One Health’ approach. This analysis can involve direct analysis of food for pathogens, using a combination of traditional and molecular methods, or application of molecular methods to characterisation of pathogenic bacteria isolated. Molecular methods offer many advantages in terms of specificity, sensitivity (under certain conditions), time-to-result and in the characterisation of isolates, for example with whole genome sequencing. However, it is important to be aware of the current limitations of molecular methods used in food analysis. In this review, the advantages and challenges of molecular methods for the analysis of food and of pathogens isolated from food associated environments are discussed with particular focus on the opportunities and current limitations of such methods.