One hundred and fifteen adults aged 60–75 years with knee osteoarthritis were randomized in a double-blind clinical trial to receive multistrain probiotics or a placebo for 16 weeks. During the follow-up, 14 patients discontinued probiotic treatment and 6 patients discontinued placebo, resulting in 95 patients included (46 taking probiotics, 49 taking the placebo). The primary outcomes of interest were pain intensity measured by visual analog scale and functional capacity measured by the Short Physical Performance Battery (SPPB). Secondary outcomes included Oxford Knee Score (OKS), knee flexion range of motion, gait speed, handgrip strength, and biochemical markers of intestinal permeability (zonulin), c-reactive protein (CRP), and oxidative stress (8-isoprostanes). Outcomes were assessed at baseline and 16 weeks. Overall, the results of the study revealed that probiotic supplementation significantly improved functional performance, pain during walking, knee range of motion, gait speed, and handgrip strength compared with placebo, accompanied by reductions in systemic inflammation and intestinal permeability. These findings suggest that probiotics may serve as an effective addition as a non-pharmacologic therapy for improving physical function in knee osteoarthritis.