Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 are two strains frequently used as probiotic components in food supplements. The decrease of potentially pathogenic gastrointestinal microorganisms is one of their claimed mechanisms. The aim of this study was to investigate their ability, alone or in combination, to inhibit in vitro the growth of Gram-negative, Gram-positive and Candida reference strains and clinical isolates, using different methods. The cell-free supernatants were obtained by centrifugation and filtration from single or mixed broth cultures and the inhibitory activity was tested using both agar-well diffusion and broth microdilution methods. In order to get some preliminary information about the chemical nature of the active metabolites released in the supernatants, the inhibitory activity was investigated after neutralization, heat and proteolytic treatments. The highest inhibitory activity was shown by the untreated supernatant obtained from broth culture of the two probiotic strains, especially against bacterial reference strains and clinical isolates. This supernatant showed inhibitory activity towards Candida species, too. A decreased inhibitory activity was observed for the supernatants obtained from single cultures and after proteolytic treatment, against bacterial reference strains. The study suggests that the combination of B. longum BB536 and L. rhamnosus HN001 could represent a possible alternative against gastrointestinal and urinary pathogens either as prophylaxis or as treatment.