Pattern recognition receptor (PRR) agonists are promising for use in modulating immune responses in clinical settings characterized by immune immaturity or deficiency. β-Glucans derived from Ganoderma lucidum have demonstrated immune-modulatory activity both in vitro and in vivo. To evaluate the immunomodulatory activity of orally administered β-glucans, a randomized, double-blinded, placebo-controlled clinical study was performed in asymptomatic children, aged 3 to 5 years old, from Medellin, Colombia. Primary outcomes were the circulating CD8+ T lymphocyte and natural killer cell counts; secondary outcomes were circulating lymphocyte counts (total, CD3+, and CD4+ T cells), serum concentrations of total immunoglobulin A and cytokines, and various hematological parameters. The treatments were administered daily for 12 weeks, and physical and laboratory evaluations were performed at days 0 and 84. Children in the group receiving a yogurt with β-glucans presented a significantly higher absolute count of peripheral blood total lymphocytes (CD3+, CD4+, and CD8+ T cells) than that in the group receiving placebo. The interventions were safe and well tolerated; no abnormal increases in serum creatinine or hepatic aminotransferases occurred, and adherence was higher than 90% in the intervention groups. This study demonstrates that β-glucans from G. lucidum increase the frequency of immune system cells in the peripheral blood; these cells are critical in the defense against infectious threats in asymptomatic children 3 to 5 years old. These findings warrant longer controlled clinical trials that aim to evaluate the efficacy of β-glucans in preventing infections in healthy children and to define their potential to enhance lymphoid cell number and functions in various lymphoid immune deficiencies.