Previous inventories of the diversity of lepidopteran symbionts have been limited to Eubacteria. We conducted the first comprehensive microbial survey of a lepidopteran insect, Calyptra thalictri Borkhausen (Lepidoptera: Noctuidae), using polymerase chain reaction (PCR) primers for 16S rRNA sequences for Eubacteria, and primers for Archaea, Fungi, Microsporidia, Wolbachia, and yeast-like organisms. In an effort to better understand the biology and vector capabilities of vampire moths, the survey was conducted using the head and abdomens separately of adult males of the fruit-piercing and blood-feeding moth C. thalictri. High-fidelity PCR and subsequent DNA analyses indicated that at least five microorganisms belonging to the alpha, beta, and gamma-Proteobacteria are associated with C. thalictri. Two eubacterial sequences, related to a Klebsiella sp. and a Sinorhizobium sp., were detected in the abdomens of all individuals sampled, and three additional sequences, two related to species in the genus Alcaligenes, and one related to a Rhizobium sp., were found in some of the abdominal samples, suggesting all five could be gut symbionts. No Archaea, Fungi, Microsporidia, Wolbachia, or yeast-like organisms were detected. These results document the first case of endosymbiotic bacteria detected in a fruit-piercing and blood-feeding moth. The possible role these microorganisms play in the biology of C. thalictri is discussed.