The long-term functional adaptation of innate immune cells following an initial stimulation, referred to as trained immunity or innate immune memory, enhances responsiveness and protection against secondary infections. Toll-like receptors (TLRs), an evolutionarily conserved family, recognize microbial-associated molecular patterns, initiating innate and adaptive immune responses. TLR signaling cascades induce the production of pro-inflammatory cytokines, antimicrobial peptides, and interferons, promoting pathogen clearance, while also driving epigenetic and metabolic reprogramming that enhances immune responses and protection to subsequent challenges. However, TLRs also recognize endogenous ligands contributing to chronic inflammation and autoimmune diseases. This review examines the role of TLRs and their various agonists in mediating trained immunity across diverse immune cell types, with an emphasis on their dual role in protecting against infections and chronic inflammation. It highlights recent clinical trials of TLR agonists as immunomodulatory agents and their therapeutic potential in infectious diseases and cancer. By providing an in-depth analysis of TLR-driven trained immunity, this review highlights the extensive influence of TLRs on immune cell populations and their implications for the development of novel, broad-spectrum immunotherapies.