The presence of antibiotic resistance genes (ARGs) in wastewater and their dissemination into aquatic ecosystems represents a significant threat to ecological integrity and public health. This study focuses on the Batuco Lagoon Natural Sanctuary (receiving effluents from a WWTP) and on irrigation canals used for agriculture (without WWTP influence). The aim was to analyze seasonal ARG dynamics and relate their occurrence to water physicochemical characteristics. Five sampling campaigns were conducted, during which physicochemical analyses and molecular detection were performed to identify the blaTEM, sul1, tetW, and ermB genes. Dissolved oxygen ranged from 2.8 to 5.0 mg/L, COD in untreated wastewater reached 287.84 mg/L, and the lagoon exhibited hypereutrophic conditions with total nitrogen averaging 4.56 mg/L (BL1) and 4.39 mg/L (BL2). blaTEM was the most prevalent gene, detected in 71% of samples, followed by sul1 (63%) and tetW (50%). ermB was the least prevalent, present in only 25% of samples. Significant seasonal variations were observed: 33% of samples were ARG-positive in winter, 38% in spring, 50% in fall, and 88% in summer, with all four genes detected during the latter. Seasonal factors, especially summer temperatures and human activity, may increase ARG dissemination, highlighting the need for better WWTP effluent management.