In this study, biofilm was grown on stainless steel surfaces (AISI 304) for 2, 5, and 10 d at 25°C in slime broth inoculated with the food spoilage microbes Bacillus subtilis, Listeria monocytogenes, Pediococcus pentosaceus, and Pseudomonas fragi. The biofilm developing on steel surfaces were investigated using conventional plating, quantitative glycocalyx determination, and epifluorescence microscopy with image analysis. The results showed that B. subtilis and P. fragi could easily be cultivated after 2 d growth. After a growth period of 10 d, the cells were difficult to cultivate from the surface, and the growth was detected better by microscopy. L. monocytogenes, on the other hand, could easily be detected by cultivation after 2, 5, and 10 d. The greatest amount of slime was produced by P. pentosaceus, as was also shown by epifluorescence microscopy.