Background: The purpose of the present investigation was to characterize the effect of probiotics on the composition of the salivary microbiota and salivary levels of inflammation-related proteins during short-term sugar stress. We tested the hypotheses that consumption of probiotics may partly counteract the detrimental influence of sugar stress on oral homeostasis. Methods: The present study was a five-week, blinded, randomized controlled trial with four study arms-A: sucrose and probiotic (n = 20); B: sucrose and placebo (n = 20); C: xylitol and probiotic (n = 20); D: xylitol and placebo (n = 20). Saliva samples were collected at baseline and after two and five weeks. The salivary microbiota was characterized by means of 16S rDNA sequencing, and sequences were referenced against the Human Oral Microbiome Database (HOMD). Neutrophil gelatinase-associated lipocalin (NGAL) and transferrin levels were quantified using immunoassays. Results: Sugar stress induced a significant increase in the relative abundance of the genus Streptococcus from 29.8% at baseline to 42.9% after two weeks. Changes were transient and were completely reversed three weeks after discontinuation of sugar stress. Xylitol and probiotics alone had no effect on the salivary microbiota, whereas the combination of xylitol and probiotics induced a significant decrease in the relative abundance of Streptococcus species from 37.6% at baseline to 23.0% at week 2. Sugar stress significantly increased salivary transferrin levels, and the effect was partly counteracted by concomitant use of probiotics. Conclusions: The data clearly demonstrate an impact of combined consumption of xylitol and probiotics on the composition of the salivary microbiota. Future studies are needed to evaluate whether the combined use of xylitol and the probiotic strains tested could have clinically protective effects during periods of sugar stress.