During cheese processing and ripening, bacteria develop as colonies. Substrates and metabolites must then diffuse either from or into the colonies. Exploring how the inner cells of the colony access the substrates or get rid of the products leads to study the permeability of bacterial colonies in cheese. Indeed, if diffusion limitation exists, the inner cells may be starved and may not be in the same physiological state than the outer cells of the colony. The question is the same for the diffusion of intracellular proteolytic enzymes released after bacterial lysis during ripening, which has never been studied in cheese. The objective of this study was to explore whether Lactococcus colonies in a model cheese were permeable to the diffusion of molecules. We varied size, shape and charge of diffusing molecules in order to explain how permeability was influenced. We used fluorescent-labelled molecules and time-lapse confocal microscopy examinations.We showed that colonies were permeable to flexible and neutral-charged dextrans whatever their size (from 4 to 155 kDa), but were not permeable to globular and charged milk proteins.We suspect the surface properties of bacterial cells (hydrophobicity and surface charges) to be of great importance on the permeability of the colony.