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<i>Streptococcus salivarius</i> (<i>S. salivarius</i>) K12 supplementation has been found to reduce the risk of recurrent upper respiratory tract infections. Yet, studies have not reported the effect of supplementation on oral <i>S. salivarius</i> K12 levels or the salivary microbiome. This clinical trial was designed to determine how supplementation with <i>S. salivarius</i> K12 influences the oral microbiome. In a randomized, double-blind, placebo-controlled trial, 13 healthy adults received a probiotic powder (PRO) containing <i>Lactobacillus acidophilus</i>, <i>Bifidobacterium lactis</i>, and <i>S. salivarius</i> K12 and 12 healthy adults received a placebo-control powder (CON) (<i>n</i> = 12) for 14 consecutive days. Oral <i>S. salivarius</i> K12 and total bacteria were quantified by qPCR and the overall oral microbiome was measured using 16S rRNA amplicon sequencing. Supplementation significantly increased mean salivary <i>S. salivarius</i> K12 levels by 5 logs compared to baseline for the PRO group (<i>p</i> < 0.0005), which returned to baseline 2 weeks post-supplementation. Compared with the CON group, salivary <i>S. salivarius</i> K12 was 5 logs higher in the PRO group at the end of the supplementation period (<i>p</i> < 0.001). Neither time nor supplementation influenced the overall oral microbiome. Supplementation with a probiotic cocktail containing <i>S. salivarius</i> K12 for two weeks significantly increased levels of salivary <i>S. salivarius</i> K12.