Kyllinga nemoralis also known as, whitehead spike sedge is a perennial herb which contains antiviral, antibacterial, antioxidant and anti-bleeding properties. This study was designed to determine the biological activity of K. nemoralis methanolic roots extract on herpes simplex virus type 1 (HSV-1) replication cycle. The effect on HSV-1 replication phases was observed by performing time-of-addition and time removal assays. Meanwhile, the level of HSV-1 selected genes expression was analysed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). In the time addition assay, K. nemoralis extract anti-HSV-1 activity was found to be most optimum when given at 2 hpi (>45% plaque reduction). The time removal assay showed that >80% plaque reduction was achieved when treatment of K. nemoralis extract was given until 24 hpi. K. nemoralis extract suppressed immediate early, early and late phases of HSV-1 replication cycle by altering the expression of UL54, UL27 and UL30 genes during the infection. This study showed that K. nemoralis methanolic roots extract has potential as anti HSV-1 by reducing the expression of HSV-1 genes at different phases of viral replication.