Adenoviruses (AdVs) are prevalent and give rise to chronic and re-current disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and-B14, cause respiratory disease and constitute a health threat for immunocom-promised individuals. HAdV-Cs are well known for lysing cells owing to the E3CR1--encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor ofHAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingre-dient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus(HIV) aspartyl protease that is used to treat AIDS. It is not effective againstsingle-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-freetransmission of HAdV, indicated by the suppression of comet-shaped infectionfoci in cell culture. Comet-shaped foci occur upon convection-based transmissionof cell-free viral particles from an infected cell to neighboring uninfected cells.HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shapedfoci, indicating that ADP enhances but is not required for cell lysis. This was sup-ported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensi-tive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and-D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growinground HAdV-C2 foci, independent of neutralizing antibodies in the medium, in-dicative of nonlytic cell-to-cell transmission. Our study demonstrates the repur-posing potential of nelfinavir with postexposure efficacy against different HAdVsand describes an alternative nonlytic cell-to-cell transmission mode of HAdV.