Endolysosomal compartments act as critical sorting hubs for viral entry, trafficking, and uncoating, yet the contribution of lipid-modifying enzymes to these processes remains incompletely understood. Acid sphingomyelinase (ASM), a key regulator of endolysosomal membrane composition, has been implicated in host-pathogen interactions, and is actively engaged in human adenovirus (HAdV) infection. Here, we confirm ASM activity as an essential host determinant of efficient HAdV infection and demonstrate that its pharmacological inhibition synergizes with direct-acting antiviral therapy. Analysis of publicly available human datasets revealed age-associated changes in expression of host cell determinants for HAdV entry like ASM expression in respiratory epithelial tissues and colonic tissue, suggesting that ASM-dependent endolysosomal functions are modulated across the human lifespan. Using epithelial cell models of HAdV infection, we show that viral entry and replication critically depend on ASM activity. Pharmacological inhibition of ASM by functional inhibitors such as fluoxetine resulted in a dose-dependent suppression of viral replication and significantly reduced infection rates in single-cycle infection assays, indicating disruption of early stages of the viral life cycle. Mechanistically, ASM inhibition impaired HAdV uptake and trafficking by reducing viral internalization and co-localization with early endosomes. Importantly, combined treatment with fluoxetine and the viral DNA polymerase inhibitor brincidofovir acts synergistically in reducing HAdV titer. These findings demonstrate that targeting host-controlled endosomal entry pathways can potentiate the antiviral efficacy of direct-acting agents. Together, our study validates ASM as a central regulator of HAdV entry and identifies combined host- and virus-directed therapy as a promising strategy to suppress HAdV infection.