Commercial vaccines against human papillomavirus (HPV) have low uptake due to parental autonomy, dosing regimen, cost, and cold chain storage requirements. Carrageenan (CG)-based formulations prevent HPV infection in vitro and in vivo but data are needed on the durability of anti-HPV activity and the effect of seminal plasma (SP). The Population Council's PC-515 gel and the lubricant Divine 9 were tested for their physicochemical properties and anti-HPV activity against HPV16, 18, and 45 pseudoviruses (PsVs). Anti-PsV activity was estimated using the luciferase assay in HeLa cells and the HPV PsV luciferase mouse model. Formulations were applied intravaginally either 2h pre/2h post (-2h/+2h) or 24h pre (-24h) relative to challenge with HPV16 or 45 PsV in PBS or SP/PBS. Both formulations showed broad-spectrum anti-HPV activity in vitro (IC50 1-20ng/ml), significantly decreasing HPV PsV infection in the mouse model (-2h/+2h, p<0.0001). PC-515 protected better than Divine 9 in the -24h dosing regimen (p<0.0001) and comparable to Divine 9 in the -2h/+2h regimen (p=0.9841). PC-515 retained full activity in the murine model when PsV solutions contained human SP. The durable, potential broad-spectrum anti-HPV activity of CG formulations in the presence of SP supports their further development to prevent HPV acquisition.