Iota-carrageenan (IC) nasal spray, a medical device approved for treating respiratory viral infections, has previously been shown to inhibit the ability of a variety of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to enter and replicate in the cell by interfering with the virus binding to the cell surface. The aim of this study was to further investigate the efficacy and safety of IC in SARS-CoV-2 infection in advanced in vitro models of the human respiratory epithelium, the primary target and entry port for SARS-CoV-2. We extended the in vitro safety assessment of nebulized IC in a 3-dimensional model of reconstituted human bronchial epithelium, and we demonstrated the efficacy of IC in protecting reconstituted nasal epithelium against viral infection and replication of a patient-derived SARS-CoV-2 strain. The results obtained from these two advanced models of human respiratory tract epithelia confirm previous findings from in vitro SARS-CoV-2 infection assays and demonstrate that topically applied IC can effectively prevent SARS-CoV-2 infection and replication. Moreover, the absence of toxicity and functional and structural impairment of the mucociliary epithelium demonstrates that the nebulized IC is well tolerated.