Baia da Ilha Grande (BIG), Rio de Janeiro, Brazil, is one of the largest bays in the world. BIG is important because it serves as a route for the mining and oil industries and plays a vital role in mariculture activities. However, BIG has suffered significant impacts in recent years due to increased pollution and climate change, culminating in a local mariculture collapse. We examined the pollution levels of the bay. Biogeochemical, microbiological, and metagenomics analyses were conducted in ten rivers during the 2022 dry and rainy seasons. Combined data analyses showed that the bay's ten most significant rivers are polluted and classified into three decreasing levels of pollution groups (P1-P3). The P1 group (Centro, Japuíba, Jacuecanga) had the worst-case scenario for all pollution types, and the highest number of the nearby populations, nautical workshops and hospitals. Whereas the P2 (Jacarei, Perequeaçu and Taquari) and P3 (Frade, Bracuí, Mambucaba, São Roque) had relatively reduced pollution, as shown mainly by fecal bacteria. Metals, such as Al (>
0.3 mg/L), Fe (>
1.4 mg/L), Pb (>
0.15 mg/L), and resistance genes (∼2 % metagenomic profile) were also more abundant in P1. High levels of metals and antibiotic resistance genes were a strong indication of pollution. The results from this study shed light on the health status of BIG rivers for further conservation programs and public policies to prevent rivers and marine biodiversity losses, and they serves as a warning on the urgent need to treat effluents in the region.