Background and objective <p>Gut microbiota dysbiosis, a common complication in mechanically ventilated (MV) patients requiring intensive care, contributes to systemic inflammation and immune dysfunction. Probiotics have been demonstrated to modulate gut microbiota composition, improve intestinal mucosal integrity, and affect short-chain fatty acid (SCFA) production and metabolic pathways. This research examines the effects of oral probiotics on serum non-targeted metabolomics and SCFA production in these patients, with a specific focus on elucidating the mechanisms by which probiotics enhance mucosal immune function.</p> Methods <p>This prospective pilot study enrolled ten mechanically ventilated patients who received add-on therapy with oral probiotics alongside routine treatment. Serum samples were collected before and after 14 days of treatment to measure procalcitonin (PCT), IL-6, IL-17, and IgA levels, while fecal samples were analyzed for the concentrations of three key SCFAs (acetic acid, propionic acid, and butyric acid). Changes in serum non-targeted metabolomics were assessed using ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS).</p> Results <p>After probiotic therapy, the levels of PCT and IL-17 in the serum of patients were significantly reduced compared to before treatment, while IgA concentration significantly increased (<i>P</i> &lt; 0.05), but the concentrations of the three short-chain fatty acids in the feces did not change significantly (<i>P</i> &gt; 0.05). Probiotic therapy significantly affected the metabolism of arachidonic acid, glycerophospholipids, and tryptophan in patients.</p> Conclusion <p>Oral probiotics reduce inflammation, increase IgA levels, and influence serum metabolomics. Their effects may be mediated through arachidonic acid, glycerophospholipid, and tryptophan metabolism pathways.</p>