Hydroxypropyl-beta-cyclodextrin (HP-BCD) is a cholesterol sequestering agent that has been explored as a potential antiviral drug against enveloped viruses and is currently under clinical investigation for Niemann Pick C disease. We previously demonstrated that HP-BCD inhibits SARS-CoV-2 replication and infectivity by targeting both host cells and viral particles. In this study, we investigated the molecular mechanisms underlying HP-BCD antiviral activity against SARS-CoV-2 in vitro. Treatment of Vero cells with HP-BCD altered intracellular compartmentalization, as indicated by the redistribution of early endosome markers. Also, HP-BCD treatment prior to SARS-CoV-2 inoculation promoted an accumulation of nucleocapsid protein at early time points after infection. Infected cells pretreated with HP-BCD exhibited reduced cholesterol and lipid droplets content, potentially hampering the formation of new double-membrane vesicles. Consistently, viral replication complexes were scarcely detected in treated cells, which displayed disformed vesicle-like structures and lacked membrane-associated virus particles. Notably, HP-BCD also reduced viral replication when administered after virus adsorption, although with lower efficacy, indicating interference with potentially multiple stages of the SARS-CoV-2 life cycle. These findings suggest that HP-BCD effectively impairs SARS-CoV-2 biosynthesis through multiple mechanisms with minimal cytotoxicity, supporting its potential as a promising antiviral candidate.