This study explores the potential spillback of dengue virus (DENV) from urban to wild environments through the detection of neutralizing antibodies against DENV in sylvatic populations of bats from the Argentinean Yungas. Sampling campaigns were conducted in the Yungas biogeographical region of northwest Argentina during April 2021 and 2022. Bat blood samples were collected and tested for neutralizing antibodies against DENV-1 using the Plaque-Reduction Neutralization Test (PRNT). DENV-1 antibodies were detected in 2.8% (4/143) of the bat samples, with positive samples found in four species: Sturnira lilium, S. erythromos, Artibeus planirostris, and Tadarida brasiliensis. All from sylvatic habitats. The presence of DENV-1 antibodies in sylvatic bats suggests potential exposure to the virus in wild areas, indicating a possible spillback of DENV in subtropical areas of Argentina. Further research is needed to understand the role of bats as reservoirs for DENV and their impact on the virus endemicity.