Dengue fever is an acute febrile disease caused by dengue virus (DENV) infection. Over the past 60 years, DENV has spread throughout tropical regions and currently affects more than 50% of the world’s population; however, there are as of yet no effective anti-DENV drugs for clinical treatment. A number of research teams have investigated derivatives of glycolipids as possible agents for the inhibition of DENV. Our objective in this research was to study the antiviral effects of trehalose 6-caprate (TMC), trehalose 6-monolaurate (TML), and trehalose 6-monooleate (TMO), based on reports that the corresponding glycosyl, trehalose, reduces the transmission of Zika virus (ZIKV). We also sought to elucidate the molecular mechanisms underlying inhibition using the RNA isolation and reverse transcription-quantitative polymerase chain reaction, western blot analysis, median tissue culture infectious dose (TCID ) assay, and immunofluorescence assay and immunochemistry staining, in vitro. This is the first study to demonstrate the TML-induced inhibition of DENV serotype 2 (DENV-2) in a dose-dependent manner. The inhibitory effects of TML in the pretreated, cotreated, and full-treated groups were confirmed using time of addition assays. We determined that TML restricted viral binding, entry, replication, and release. We also confirmed the efficacy of TML against three clinical isolates of DENV serotypes 1, 3, and 4 (DENV-1, DENV-3, and DENV-4). The findings obtained in this study identify TML as a promising candidate for the development of drugs to treat DENV infection.© 2023 Wiley Periodicals LLC.