Chlorophyll fluorescence imaging is a non-invasive, non-destructive means with which to examine the impact of fungal pathogens on the photosynthetic metabolism of host plants. As such, it has great potential for screening purposes in high-throughput phenomics environments. However, there is great diversity in the responses of plants to different plant-fungal pathogens and the choice of suitable experimental conditions and protocols and interpretation of the results requires both preliminary laboratory experiments and an understanding of the biology of the specific plant-pathogen interaction. In this review, we examine the interaction between biotrophic, hemi-biotrophic and necrotrophic fungal pathogens and their hosts to illustrate the extent to which chlorophyll fluorescence imaging can be used to detect the presence of disease before the appearance of visible symptoms, distinguish between compatible and incompatible fungal interactions, identify heterogeneity in photosynthetic performance within the infected leaf and provide insights into the underlying mechanisms. The limitations and challenges of using chlorophyll fluorescence imaging in high throughput screens is discussed.