Marathon runners, subjected to intense training regimes and prolonged, exhaustive exercises, often experience a compromised immune response. Probiotic supplementation has emerged as a potential remedy to mitigate the impact of prolonged exercise on athletes. Consequently, this study sought to assess the influence of probiotic supplementation on monocyte functionality both before and after the official marathon race. Twenty-seven runners were randomly and double-blindly assigned to two groups: Placebo-PLA (n=13) and Probiotic-PRO (n=14). Over thirty days, both groups received supplements—PLA sachets containing maltodextrin (5g/day) and PRO sachets containing 1 x 1010 CFU Lactobacillus acidophilus and 1 x 1010 CFU Bifidobacterium bifidum subsp. lactis. Blood samples were collected, and immunological assays, including phagocytosis, hydrogen peroxide production, cytokine levels, and monocyte immunophenotyping, were conducted at four different intervals: Baseline (start of supplementation/thirty days pre-marathon), 24h-Before (one-day pre-marathon), 1h-After (1h post-marathon), and 5d-After (five days post-marathon). Monocyte populations remained consistent throughout the study. A notable increase in phagocytosis was observed in the PRO group after thirty days of supplementation. Upon LPS stimulation, both PRO and PLA groups exhibited decreased IL-8 production. However, after the marathon race, IL-15 stimulation demonstrated increased levels of 5d-After, while IL-1-β, IL-8, IL-10, IL-15, and TNF-α varied across different intervals, specifically within the PRO group. Probiotic supplementation notably enhanced the phagocytic capacity of monocytes. However, these effects were not sustained post-marathon.