The Alphavirus chikungunya (CHIKV) is an arbovirus that belongs to the Togaviridae family and causes Chikungunya Fever (CHIKF). As a member of the Alphavirus genus, CHIKV is an arthritogenic virus that causes severe polyarthritis and myalgia along with symptoms like fever, rash and headaches. Following the Kenyan epidemic in 2004, CHIKV has spread to the Indian Ocean Islands and reached Europe, drawing global attention. In 2014, CHIKV reached South America, establishing reemergent outbreaks. Anti-inflammatory drugs and analgesics are the mainstay of treatment for symptomatic relief in infected patients. However, no clinically approved virus-specific drugs are available for CHIKV infection. In this context, plant-derived compounds arise as alternatives for drug research, given its large availability in nature. Our research group has already demonstrated that a glycosylated lignan (GL) from Phyllanthus brasiliensis has antiviral activity against CHIKV and other arboviruses. Therefore, in aiming to describe the GL mechanism of action, we conducted a series of experiments, targeting different moments of the viral infection cycle. First, we evaluated whether the GL can affect the structure of CHIKV particles and their ability to attach cell membranes through a virucidal and adsorption assay, respectively. Both experiments did not demonstrate significance. Thereafter, we conducted an internalization and post-infection assay to measure if the GL could inhibit viral entry and intracellular steps of the CHIKV infectious cycle. Both assays demonstrated significance (p<0,05). Considering that the GL reduced the CHIKV titer in the initial hours after compound addition, we propose that GL acts in the initial steps in the CHIKV infectious cycle, comprising stages between endocytosis and viral replication. These results demonstrate the ability of GL from P. brasiliensis to inhibit CHIKV replication, hinting at its potential to be used as a starting scaffold for further development of antivirals against CHIKF.