Modulation of the immune response is an established feature of certain lactobacilli. CXCL8 is an inflammatory chemokine released by the urinary tract mucosa after contact with uropathogenic Escherichia coli during urinary tract infection and is crucial for proper infiltration of immune cells. Nevertheless, persistently high levels of CXCL8 are associated with pathogenicity and malignancy. In this study, we tested twelve Lactobacillus strains for their ability to influence CXCL8 release from urothelial cells. We evaluated how strains from different Lactobacillus species could regulate CXCL8 in human 5637 urothelial cells, either resting cells or cells concomitantly challenged with heat-killed E. coli. A majority of the tested species altered CXCL8 release from the urothelial cells after 24 hours of stimulation. Most species increased CXCL8 release, whereas a few lactobacilli efficiently suppressed CXCL8 secretion from E. coli-challenged cells. While strong CXCL8 modulators such as Lactobacillus reuteri and Lactobacillus delbrueckii were unable to degrade CXCL8 in the extracellular environment, effects on IL8 transcription were evident for selected lactobacilli. Although IL8 transcription was affected by lactobacilli, the influence on mRNA transcript did not correlate to the impact on CXCL8 release. Phylogenetic analysis based on a 16S rRNA dendrogram of the tested lactobacilli and their effect on CXCL8 revealed some linkage to specific Lactobacillus groups. Testing the immunomodulatory nature of lactobacilli can prove important when selecting new probiotic microbes. Moreover, we believe that phylogenetic and phenotypic similarities could be used to analyse the traits governing such modulation.