Human cytomegalovirus (HCMV) causes a life-long infection and can affect up to 90% of the population depending on location and age. Even though the infection is predominantly asymptomatic in healthy individuals, immunocompromised patients such as HIV-positive patients and organ transplant recipients can develop serious and fatal disease. Additionally, HCMV infection during pregnancy is one of the main causes of virus-associated birth defects. Protective measures against HCMV are limited, due to the lack of vaccines and the toxicity of and emerging resistance to antiviral treatments. Whilst the early stages of viral replication are well characterised, less is known about the late stages, and the process by which infectious virions are assembled and released from infected cells. However, a key indicator of late-stage infection is the biogenesis of the cytoplasmic megastructure known as the viral assembly complex (vAC). vAC architecture consists of endosome-derived vesicles centred within concentric rings of the trans- and cis-Golgi network and it is understood to be the site for secondary envelopment of maturing virions. Using a library of HCMV mutants (>100), we screened viruses for their competence to spread and ability to form a vAC. We discovered a subset of viral mutants that displayed typical vACs, yet were unable to complete the viral life cycle, indicating a likely role in either envelopment, trafficking, membrane fusion, or re-infection. From this subset, we characterised a crucial viral protein and demonstrate that its deletion from the viral genome completely abrogates the spread of HCMV, but ectopic expression in host cells can rescue this phenotype. In conclusion, we have identified a novel set of proteins, which can be targeted to block HCMV spread and inhibit viral egress as a novel antiviral axis.