As the population ages, cognitive decline becomes more common. Strategies targeting the gut-brain axis using probiotics are emerging to achieve improvements in neuropsychiatric and neurological disorders. However, the beneficial role of probiotics on brain function in healthy older adults remains unclear. Our aim was to evaluate a multi-species probiotic formulation as a therapeutic approach to reduce emotional and cognitive decline associated with aging in healthy adults. A randomized double-blind placebo-controlled crossover trial was conducted. The study involved a 10-week intervention where participants consumed the assigned probiotic product daily, followed by a 4-week washout period before the second condition started. Cognitive function was assessed using the Mini-Mental State Examination (MMSE) and the Psychological Experiments Construction Language Test Battery. At the emotional level, the Beck Depression Inventory (BDI) and the State-Trait Anxiety Inventory (STAI) were used. Thirty-three participants, recruited between July 2020 and April 2022, ingested a multispecies probiotic (Lactobacillus rhamnosus and Bifidobacterium lactis). After the intervention, noticeable enhancements were observed in cognitive function (mean difference 1.90, 95% CI 1.09 to 2.70, p < 0.005), memory (mean difference 4.60, 95% CI 2.91 to 6.29, p < 0.005) by MMSE and digit task, and depressive symptoms (mean difference 4.09, 95% CI 1.70 to 6.48, p < 0.005) by BDI. Furthermore, there were significant improvements observed in planning and problem-solving skills, selective attention, cognitive flexibility, impulsivity, and inhibitory ability. Probiotics administration improved cognitive and emotional function in older adults. Limited research supports this, requiring more scientific evidence for probiotics as an effective therapy for cognitive decline. This study has been prospectively registered at ClinicalTrials.gov (NCT04828421; 2020/July/17).