Previously, we have shown that facultative pathogens Serratia grimesii and Serratia proteamaculans are capable to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin or protealysin, respectively (Bozhokina et al. in Cell Biol Int 35(2):111-118, 2011). Noninvasive Escherichia coli transformed with grimelysin or protealysin gene became invasive, indicating that the protease is a virulence factor. Here we elucidated involvement of other virulence factors in the invasion of S. grimesii and S. proteamaculans. Under similar experimental conditions, the amount of S. proteamaculans internalized within human carcinoma HeLa cells was fivefold higher than that of S. grimesii. In accord with this, in S. proteamaculans, high activities of pore-forming hemolysin ShlA and extracellular metalloprotease serralysin were detected. In S. grimesii, activity of toxin ShlA was not detected, and the serralysin activity of the bacterial growth medium was very low. We also show that iron depletion strongly enhanced invasive activity of S. proteamaculans, increasing activities of hemolysin ShlA and serralysin, but did not affect S. grimesii properties. These results show that the invasive activity of S. proteamaculans is maintained, along with protealysin, by hemolysin and serralysin. On the other hand, grimelysin is so far the only known invasion factor of S. grimesii.