Biofilms of the Gram-negative bacteria Pseudomonas aeruginosa and Pseudomonas fragi were grown on stainless steel surfaces (AISI 304, 2B) for 4 days in slime broth. These biofilms were treated with four commercial disinfectants. The disinfectants were alcohol-based, tenside-based, peroxide-based and chlorine-based products, covering most disinfectant types used in the food industry. The effects of the disinfectants on the bacterial cells were first investigated in suspension using the permeabilisation test, which is based on fluorescence assessment of hydrophobic 1-N-phenyl-naphtylamine (NPN). The surfaces covered with disinfectant-treated biofilms were investigated using conventional cultivation, impedimetry and epifluorescence microscopy in combination with image analysis of preparations stained with the DNA-stain acridine orange and with the metabolic indicator system CTC-DAPI. The results showed that the tenside-based and peroxide-based disinfectants permeabilised the cells in suspension. The overall biofilm results showed that of the agents tested, the peroxide-based and chlorine-based disinfectants acted most effectively on cells in biofilms.