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The role of probiotic supplementation in type 2 diabetes (T2D) treatment is controversial. The present study aimed to assess the effects of a multi-strain probiotic supplement (LactoLevure^R (containing <i>Lactobacillus acidophilus</i>, <i>Lactobacillus plantarum</i>, <i>Bifidobacterium lactis</i>, and <i>Saccharomyces boulardii</i>)) over 6 months, primarily on glycemic control as well as on lipid levels and alterations in the gut microbiome, among individuals with T2D residing in Greece. A total of 91 adults with T2D (mean age [±SD] 65.12 ± 10.92 years, 62.6% males) were randomized to receive the probiotic supplement or a matching placebo capsule, once daily, for 6 months. Blood chemistries and anthropometric parameters were conducted every 3 months, and stool samples were collected at baseline and at 6 months. Significant reductions in HbA1c, fasting blood glucose, and total cholesterol were observed in participants treated with the probiotic supplement (n = 46) compared to the controls (n = 45), even after adjustment for a greater decrease in adiposity (waist circumference). Although there were no statistically significant differences in the diversity of the gut microbiome (α and β diversity), the administration of probiotics did influence several genera, metabolites, and key enzymes associated with diabetes. Overall, the administration of the multi-strain probiotic LactoLevure^R over a 6-month period in individuals with T2D was well-tolerated and had a positive impact on metabolic parameters, alongside improvements in indices of adiposity.