Pectin accounts for one of the major polysaccharides of the plant cell wall. Pectin methylesterases (PMEs) are enzymes that are able to alter the pectin structure and affect its gelling properties by de-esterifying galacturonic acid. In this work, the diffusion of two PMEs with different origins and modes of action was characterised with a multi-scale approach in different media consisting of pectin macromolecular solutions and physical gels. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescent Correlation Spectroscopy (FCS) investigations showed similar diffusion behaviour for both enzymes at a microscopic scale. In contrast, their behaviours tended to differ when increasing the observation scale up to mm. Indeed, fungal PMEs diffused faster and in a larger range than plant PMEs. These results are discussed with regard to the respective modes of action, processivity and in vivo roles of the two enzymes.