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Seagrass beds in Florida Bay are home to many ecologically and economically important species. Anthropogenic press perturbation via alterations in hydrology and pulse perturbations such as drought can lead to hypersalinity, hypoxia, and sulfide toxicity, ultimately causing seagrass die-offs. Florida Bay has undergone two large-scale seagrass die-offs, the first in the late 1980s and early 1990s and the second in 2015. Post-die-off events, samples were collected for stable isotope analysis. Using historical (1998–1999) and contemporary (2018) stable isotope data, we examine how food webs in Florida Bay have changed in response to seagrass die-off over time by measuring contributions of basal sources to energy usage and using trophic niche analysis to compare niche size and overlap. We examined three consumer species sampled in both time periods (Orthopristis chrysoptera, Lagodon rhomboides, and Eucinostomus gula) in our study. Seagrass production comprised the majority of source usage in both datasets. However, contemporary consumers had a mean increase of 18% seagrass usage and a mean decrease in epiphyte usage of 7%. The shift in trophic niche from epiphyte usage (green pathway) toward seagrass usage (brown pathway) may indicate that food web browning is occurring in Florida Bay.