Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.