Here, we sought to deepen our understanding of molecular factors underpinning lectin antiviral function. Using a pseudovirus platform, we found that CV-N and to a somewhat lesser extent GRFT were potent as infection inhibitors, while microvirin (MVN) was essentially inactive. Furthermore, using washout experiments, CV-N and GRFT were shown to irreversibly inactivate SARS-CoV-2 pseudoviruses in a dose-dependent manner. Functional assays with single glycan attachment site SARS-CoV-2 pseudovirus mutants resulted in the identification of two glycan clusters in the S1 subunit of the Spike important for CV-N inactivation of pseudovirus. Analogous clusters were found to be important for GRFT antiviral activity. The results of this work argue that multivalent engagement of glycans in S1 oligosaccharides disrupts spike conformation, causing the inactivation of SARS-CoV-2. Overall, this work helps build the foundation for defining the conformational vulnerability of the SARS-CoV-2 Spike, the capacity of lectins to hijack this vulnerability, and the potential to develop lectin inactivators for possible COVID-19 treatment.