The pelagic tunicate pyrosome,Pyrosoma atlanticum, is known for its brilliant bioluminescence, but the mechanism causing this bioluminescence has not been fully characterized. This study identifies the bacterial bioluminescent symbionts ofP. atlanticumcollected in the northern Gulf of Mexico using several methods such as light and electron microscopy, as well as molecular genetics. The bacteria are localized within the pyrosome light organs. Greater than 50% of the bacterial taxa present in the tunicate samples were the bioluminescent symbiotic bacteria Vibrionaceae as determined by utilizing current molecular genetics methodologies. A total of 396K MiSeq16S rRNA reads provided total pyrosome microbiome profiles to determine bacterial symbiont taxonomy. After comparing with the Silva rRNA database, aPhotobacteriumsp. r33-like bacterium (which we refer to as “PhotobacteriumPa-1”) matched at 99% sequence identity as the most abundant bacteria withinPyrosoma atlanticumsamples. Specifically designed 16S rRNA V4 probes for fluorescencein situhybridization (FISH) verified thePhotobacteriumPa-1 location as internally concentrated along the periphery of each dual pyrosome luminous organ. While searching for bacterialluxgenes in two tunicate samples, we also serendipitously generated a draft tunicate mitochondrial genome that can be used forPyrosoma atlanticumidentification. Scanning (SEM) and transmission (TEM) electron microscopy confirmed the presence of intracellular rod-like bacteria in the light organs. This intracellular localization of bacteria may represent bacteriocyte formation reminiscent of other invertebrates.