Background: Nasal disinfection with 0.5% povidone-iodine (PVP-I) may be a useful adjunct in the management of COVID-19. The purpose of this article is to confirm the in vitro activity of the PVP-I nasal spray against SARS-CoV-2 and whether that may translate into reduced nasal shedding in vivo. Methods: Two SARS-CoV-2 virus isolates were exposed to 0.5% PVP-nasal spray (Nasodine®) for different times in vitro, with PCR and cell culture used to assess impact on viral infectivity and RNA copies. An open label in vivo single arm pilot study of 14 subjects with positive COVID-19 PCR diagnosis was undertaken. Baseline nasal swabs were collected to quantify SARS-CoV-2 pre-treatment, followed by a single 0.5% PVP nasal spray application (1.12 mL). Nasal swabs were collected at 5, 15, and 60 minutes post-dose to assess immediate and residual impact of treatment. Results: In vitro, the nasal spray reduced infectivity by 3.5 log10 TCID50/mL (99.97%) after 15 seconds exposure and eliminated detectable viral infectivity after 60 seconds; there was no effect on viral RNA detection by PCR. In vivo, culturable virus (VOC beta/B.1.351 variant) was obtained from 6 of 14 PCRconfirmed positive subjects; in these subjects, 5 minutes after the single PVP-I dose, the mean viral titre was reduced by 65% versus baseline and by 79% versus baseline at 60 minutes post-dose. 5 of the 6 subjects (83%), had reduction or cessation of viral shedding at 5 minutes in all 6 subjects, virus titers 60 minutes post-dose were below baseline value. 0.5% PVP-I treatment didn’t interfere with the laboratory diagnosis of COVID-19 via PCR-detection of viral RNA in humans. Conclusions: 0.5% PVP-I nasal spray is rapidly virucidal to SARS-CoV-2 in vitro using exposure times consistent with nasal residence; single in vivo nasal administration reduced infectious viral titers in COVID-19 subjects with culturable virus. A single application of 0.5% PVP-I nasal spray does not interfere with PCR-mediated laboratory diagnosis of COVID-19. We are undertaking a large double blinded randomized controlled trial to confirm if repeated application of 0.5% PVP-I nasal spray over a longer period could be useful in suppressing viral shedding and transmission risk in COVID-positive patients.