Cordyceps cicadae and Ophiocordyceps sobolifera are entomopathogenic fungi often misidentified due to taxonomic ambiguity, leading to confusion in research and applications. This study compared their metabolite compositions and immunomodulatory activities using molecular identification, chemical profiling, and in vitro assays. High-performance liquid chromatography and LC-MS/MS revealed distinct signatures: C. cicadae lacked myriocin but accumulated N6-(2-hydroxyethyl)adenosine (HEA), while O. sobolifera produced abundant myriocin but no HEA. Beauvericin was enriched in wild C. cicadae, whereas adenosine was consistently present in both species, with higher levels in O. sobolifera. Cordycepin was detected only in artificially cultivated C. cicadae. Functional assays showed that C. cicadae extracts, HEA, and adenosine significantly suppressed C48/80-induced histamine release from mast cells, and ethanol extracts of C. cicadae reduced interleukin-33 (IL-33) activation. In contrast, O. sobolifera water extracts enhanced glucagon-like peptide-1 (GLP-1) secretion, while myriocin, beauvericin, and oosporein were inactive in these pathways. These findings clarify the compositional and functional divergence between the two fungi. The absence of toxic myriocin in C. cicadae supports its safer use in food and medicinal applications, with HEA emerging as a key bioactive marker. Liquid fermentation further enhances HEA levels while reducing beauvericin, suggesting its suitability for controlled production. Meanwhile, O. sobolifera may be explored for GLP-1-related benefits. Overall, this study provides a molecular and functional basis to differentiate the two species and highlights their distinct potential in health applications.