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Wastewater pollution in seagrass has been documented for decades. In areas where baseline data are lacking, such as the Mexican Caribbean, the δ15N of seagrass can provide information on status and trends. The aims of this study were to determine the spatial and temporal variation in δ15N values of T. testudinum from 2009 to 2017, to explore the nature and causes of isotopic variability, which must be fully understood, and to examine how isotopic variability is linked to wastewater on the coast. Samples of T. testudinum were collected during 2011–2017 in Cancún and from 2009 to 2017 in Mahahual, and analysed on a mass spectrometer. The δ15N values in Cancún (9.0±1.3 ‰) were significantly (P<0.005) higher than those in Mahahual (1.9±0.9 ‰). Our results reflect the continuous supply of wastewater as a source of 15N in Cancún and the atmospheric deposition of dissolved inorganic nitrogen from uncontaminated groundwater in Mahahual. Mahahual is an ideal site to continue environmental monitoring of the healthy seagrass in the southern Mexican Caribbean. Meanwhile, measures on the treatment and final disposal of wastewater in Cancun must be addressed with a great priority.