Stable isotope analysis of tissues with different turnover rates allows for a broader temporal view of a species’ feeding habits. Epidermal mucus is a rapid to medium turnover ‘tissue’ in teleost fish, but its use in elasmobranch dietary studies is unknown. In the present study, we conducted stable isotope analysis on mucus and muscle from the giant manta ray Manta birostris. Mucus d13C values were depleted and closer to surface zooplankton d13C values compared with muscle, whereas there was no significant difference in d15N values between these two tissue types. Using diet tissue discrimination factors from the literature, there was no significant difference in the expected prey profile between muscle and epidermal mucus. However, a broader range in d13C values of expected prey was shown for mucus compared with muscle. The results suggest that if M. birostris mucus is indicative of recent dietary intake, resource use during aggregative behaviour off Ecuador is broader, but with no obvious resource switching. The present study is the first example of using bulk stable isotope analysis to evaluate mucus to investigate feeding ecology in elasmobranchs. However, the time course for the change in mucus isotope signature still needs to be determined through controlled feeding studies in an aquarium setting.