Serratia marcescens strains from a dairy-producing environment were tested for their inhibitory effect on Listeria monocytogenes, Salmonella Hartford, Yersinia enterocolitica and Escherichia coli. Inhibition of foodborne pathogens was observed in the case of a non-pigmented Serratia strain, while the pigment-producing isolate was able to inhibit only Y. enterocolitica. The co-culturing study in tryptone soya broth (TSB) and milk showed that the growth of Salmonella was inhibited in the first 24 h, but later the pathogen could grow in the presence of the Serratia strain even if its cell concentration was 1000 times higher than that of Salmonella. However, we found that (1) concentrated cell-free supernatants had stronger inhibitory activity, which confirms the extracellular nature of the antagonistic compound(s). We proved that (2) protease and chitinase enzymes can take part in this mechanism, but they are not the main inhibitory compounds. The presence of prodigiosin was observed only in the case of the pigmented strain; thus, (3) we hypothesized that prodigiosin does not take part in the inhibition of the pathogens. However, (4) the combined effect of different extracellular metabolites might be attributed to the inhibitory property. Application of concentrated S. marcescens cell-free supernatant can be an effective antibacterial strategy in the food industry, mainly in the form of a bio-disinfectant on surfaces of food-processing areas.