Mushrooms have become increasingly important as a reliable food source. They have also been recognized as an important source of bioactive compounds of high nutritional and medicinal values. The nucleobases, nucleosides and nucleotides found in mushrooms play important roles in the regulation of various physiological processes in the human body via the purinergic and/or pyrimidine receptors. Cordycepin, a 3'-deoxyadenosine found in Cordyceps sinensis has received much attention as it possesses many medicinal values including anticancer properties. In this review, we provide a broad overview of the distribution of purine nucleobases (adenine and guanine); pyrimidine nucleobases (cytosine, uracil, and thymine); nucleosides (uridine, guanosine, adenosine and cytidine); as well as novel nucleosides/tides in edible and nonedible mushrooms. This review also discusses the latest research focusing on the successes, challenges, and future perspectives of the analytical methods used to determine nucleic acid constituents in mushrooms. Besides, the exotic taste and flavor of edible mushrooms are attributed to several nonvolatile and water-soluble substances, including the 5'-nucleotides. Therefore, we also discuss the total flavor 5'-nucleotides: 5'-guanosine monophosphate (5'-GMP), 5'-inosine monophosphate (5'-IMP), and 5'-xanthosine monophosphate (5'-XMP) in edible mushrooms.