Human cytomegalovirus (HCMV) can cause life-threatening diseases in neonates and immunocompromised patients. Due to multiple problems caused by the current available drugs, development of new antiviral compounds is urgently needed. In this study, we characterize the anti-HCMV spectrum and mechanism of action of the N-N'-(bis-5 nitropyrimidyl)dispirotripiperazine derivate 27 (DSTP-27). DSTP-27 exhibited strong antiviral activity against two laboratory HCMV strains with different cell tropism as well as ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates in plaque reduction assays and viral growth kinetics experiments. Interestingly, neither infectious nor noninfectious viral particles were observed by electron microscopy. Pretreatment of cell-free virus with DSTP-27 prevented virus infection. The results from time of addition assays, in which DTSP-27 was added to cells (i) before infection, (ii) during virus adsorption, or (iii) after adsorption, demonstrated an inhibitory effect on early steps of the HCMV replication cycle. This observation was confirmed by immunofluorescence as well as Western blot analysis, whereby reduced levels of the immediate early protein IE1, the processivity factor pUL44, and the tegument protein pp28 were detected. Results from attachment and penetration analyses of prechilled human embryonic lung fibroblasts revealed that virus attachment is not blocked. In addition, DSTP-27 inactivated HCMV by stable binding. Taken together, these results demonstrate that DSTP-27 (i) blocks viral penetration by interacting with the host cell and (ii) inactivates HCMV by interacting with the virus.