AbstractBiofilms are organised aggregates of bacteria that adhere to each other or surfaces. The matrix of extracellular polymeric substances that holds the cells together provides the mechanical stability of the biofilm. In this study, we have applied Brillouin microscopy, a technique that is capable of measuring mechanical properties of specimens on a micrometre scale based on the shift in frequency of light incident upon a sample due to thermal fluctuations, to investigate the micromechanical properties of an active, live Pseudomonas aeruginosa biofilm. Using this non-contact and label-free technique, we have extracted information about the internal stiffness of biofilms under continuous flow. No correlation with colony size was found when comparing the averages of Brillouin shifts of two-dimensional cross-sections of randomly selected colonies. However, when focusing on single colonies, we observed two distinct spatial patterns: in smaller colonies, stiffness increased towards their interior, indicating a more compact structure of the centre of the colony, whereas, larger (over 45 μm) colonies were found to have less stiff interiors.