Shark microbiomes are species-specific, but mechanisms (host or environmental) driving this pattern are unknown. The epidermal surface of sharks and stingrays consists of dermal denticles (small tooth-like scales) and mucus. Gills are site for gas and waste excretion in fish. Gills, like mammalian respiratory organs, are a common site for pathogen invasion and thus the microbiome is of extreme importance. Cloaca microbiomes are commonly used as an alternative to gut microbiome sampling where faecal or gut microbiome samples are unobtainable. Southern eagle rays (Myliobatis tenuicaudatus) and Port Jackson sharks (Heterodontus portusjacksoni) share similar diets and habitats, but are evolutionarily distinct. To better understand how ecology and phylogeny impact the microbiome at different body-sites, we sampled the skin (epidermal), gills (respiratory), and cloaca (digestive) microbiomes of two ecologically similar but evolutionarily distinct hosts. Each species had significantly different microbial communities (PERMANOVA p = 0.001, df = 1, F = 12.60). When nested within species, microbial communities of each body site were distinct (PERMANOVA p = 0.026, df = 6, F = 1.3812). Each body site created a unique microbial niche reflected by microbial functions. Cloaca microbioes Of the significantly different functions found in cloaca microbiomes, N-Acetylneuraminate utilization was significantly more abundant in M. tenuicaudatus than H. portusjacksoni. N-Acetylneuraminic acid is attached to proteins produced by humans in the gut. Bacteria can present host N-Acetylneuraminic acid to disguise from the immune system. Metagenomic analysis revealed distinct microbial niches at the skin, gills, and cloaca of two ecologically similar but phylogenetically distinct hosts. This suggests that both host evolution and ecology impact microbial communities at different body sites.