Pseudomonas fluorescens is a commensal bacterium present at low level in the human digestive tract that has also been reported in many clinical samples (blood, urinary tract, skin, lung, etc.) and sometimes associated with acute opportunistic infections. It has recently been found that the human β-defensin-2 can enhance the pathogenic potential of P. fluorescens. In this study, we evaluated the effect of other intestinal molecules (5HT, SP and Epi) on growth and virulence of the clinical strain P. fluorescens MFN1032. We found that P. fluorescens MFN1032 growth was not mainly affected by these factors, but several modifications in the virulence behavior of this bacterium were observed. 5HT, SP and Epi were able to modulate the motility of P. fluorescens MFN1032. 5HT and SP had an effect on pyoverdin production and IL-8 secretion, respectively. Infection of Caco-2/TC7 cells with P. fluorescens MFN1032 pretreated by SP or Epi enhanced the permeability of the monolayers and led to a partial delocalization of F-actin to the cytoplasm. These findings show that some intestinal molecules can modulate the pathogenic potential of P. fluorescens MFN1032. We can hypothesize that this dialogue between the host and the human gut microbiota may participate in health and disease.