Background <p>Metabolic dysfunction-associated steatotic liver disease (MASLD) is associated with dysbiosis of the gut microbiota. We evaluated the effect of next generation probiotics (<i>Lactobacillus delbrueckii subsp. Lactis</i> [LL001], <i>L. helveticus</i> [LH001], and <i>Pediococcus pentosaceus</i> KID7 [PPKID7]) on liver function parameters and stool microbiome in patients with MASLD.</p> Methods <p>We conducted a double-blind parallel trial of 110 patients diagnosed with MASLD. Participants were randomly assigned to four groups given three probiotics (3 capsules [9 × 10⁹ CFU]/day, <i>n</i> = 85) or placebo (<i>n</i> = 25) alongside sylimarin for 8 weeks. Clinical characteristics, serum samples, and stool samples for 16&#xa0;S rRNA gene sequencing were collected at the start and end point of the study. The primary endpoint was improvement in liver function.</p> Results <p>In the probiotic group, LL001 treatment improved alanine transaminase (87.3 ± 8.2 to 71.1 ± 6.0 U/L, <i>P</i> = 0.01) and aspartate transaminase levels (64.9 ± 4.9 to 50.0 ± 3.5 U/L, <i>P</i> &lt; 0.01), LH001 group showed body weight reduction (78.4 ± 3.0 to 77.2 ± 2.8&#xa0;kg, <i>P</i> = 0.01), and PPKID7 reduced cholesterol levels (186.1 ± 7.0 to 178.0 ± 7.9 mmol/L, <i>P</i> = 0.03). Probiotics treatment decreased the abundance of <i>Proteobacteria</i> and increased the abundance of <i>Ruminococcaceae</i> and <i>Lachnospiraceae</i> in the LL001 group. In the pre- and post-comparison of probiotic treatment at the level of the top 20 genera, a tendency was observed to decrease the genera <i>Haemohlius</i> and Ruminococcus_g2 while increasing the genus <i>Bifidobacterium</i>.</p> Conclusion <p>Eight weeks of probiotics supplementation was associated with changes in the stool microbiome and improvements in the blood biochemical parameters of MASLD.</p> Registration number of clinical trial <p>NCT04555434.</p>