The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced due to emerging variants of concern (VOCs)1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is to cleave the viral spike protein to expose the fusion peptide for cell entry5,6. Here, we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of >106 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits entry of SARS-CoV-2 VOCs, B.1.1.7, B.1.351, P.1 and B.1.617.2. Importantly, in the K18-human ACE2 transgenic mouse model of severe SARS-CoV-2 disease, we found that N-0385 affords a high level of prophylactic and therapeutic benefit following either multiple or even a single administration. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.