In the present study various combinations of metabolic indicators were used in viability studies of foodborne spoilage and pathogenic organisms. Various metabolic stains were tested in pure culture suspensions of Pseudomonas fragi and Listeria monocytogenes containing only viable cells, both viable and dead cells or only dead cells. In addition, foodborne monospecies biofilms of L. monocytogenes and P. fragi were studied. The biofilms were grown on stainless steel (AISI 304, 2B) surfaces. The results showed that both the LIVE/DEAD Bac Light Viability Kit (LIVE/DEAD) and the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)-4′,6-diamidino-2-phenylindole (DAPI) staining procedures offer a rapid, easy and reliable method for metabolic investigation of bacteria in suspension and swabbed biofilm bacteria. Assessment of replicate samples using luciferin–luciferase-based ATP measurement and conventional cultivation corroborated the results of swabbed biofilm bacteria obtained by staining. LIVE/DEAD staining cannot be used for direct staining of biofilms on surfaces, due to interference between the stain and polysaccharides of the biofilm matrix and slime. Staining by CTC-DAPI is suitable for studying viability of cells both in suspension and in biofilms attached to surfaces. The other staining procedures tested were not satisfactory or only slightly satisfactory in distinguishing between viable and dead cells.