Biofilms are complex communities of microorganisms encased in an extracellular matrix (ECM) containing biomolecules such as proteins, extracellular DNA, polysaccharides and lipids. Here we investigated biofilms formed by uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections (UTIs), to understand how ECM comprised of curli amyloid fibres and the polysaccharide cellulose contributes to antimicrobial resistance. We used single-plane illumination microscopy combined with fluorescence correlation spectroscopy (SPIM-FCS) to quantify the diffusion of different sized molecules in UPEC biofilms. This approach generated high-resolution three-dimensional diffusion coefficient maps, demonstrating that biofilms containing curli fibres, with or without cellulose, exhibit reduced and spatially heterogeneous diffusion consistent with a molecular sieving effect that limits the transport of larger molecules through the ECM. In contrast, cellulose alone had little measurable impact on diffusion. We further demonstrate these UPEC biofilms exhibit resistance to the antimicrobial peptides LL-37 and polymyxin B, in part mediated by charge-dependent reduced diffusion. This behaviour is consistent with charge-mediated interactions with the biofilm ECM that reduce antimicrobial peptide penetration and thus promote the survival of biofilm-encased cells. The combined effect of charge-based interactions and molecular sieving leads to a significantly different distribution and mobility of differently charged peptides, providing a mechanism to explain reduced activity of cationic antimicrobial peptides in curli-positive biofilms.