Toll-like receptors (TLRs) are members of a large family of evolutionarily conserved pattern recognition receptors (PRRs), which serve as key components of the innate immune system by playing a pivotal role in sensing "nonself" ligands. Endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) can recognize pathogen-derived nucleic acid and initiate an innate immune response because they react against both self- and non-self-origin nucleic acid molecules. Accordingly, both receptor agonists and antagonists are potentially useful in disparate clinical contexts and thus are globally sought after. Recent research has revealed that agonists and antagonists share an overlapping binding region. This Perspective highlights rational medicinal chemistry approaches to elucidate the structural attributes of small molecules capable of agonism or antagonism or of elegantly switching between the two. The structural evolution of different chemotypes can provide the framework for the future development of endosomal TLR agonists and antagonists.