Lymphocystiviruses cause the Lymphocystis Disease (LCD), a fish disease that affects mainly skin and fin described for the first time in 1874. These viruses have been studied since the ’60s of the last century; however, several aetiopathogenetic features of their infection have not been completely described. Recent studies have revealed the systemic spread of the virus; however, most of the studies focused on gilthead seabream (Sparus aurata) one of the most important species of Mediterranean aquaculture. Olive flounder (Paralichthys olivaceus) is one of the most important species for Asian aquaculture and lymphocystis is responsible for severe economic losses. This study has the aim to improve the aetiopathogenetic knowledge of lymphocystis in olive flounder. Particularly, wide dissemination of viral DNA has been revealed in cutaneous and visceral tissues of olive flounder during natural infection, similarly to what observed in gilthead seabream. An immuno-histochemistry protocol using novel monoclonal antibodies (MAbs) was set up to detect viral proteins and used to immunolabel viral antigens inside the infected skin cells. The use of two MAbs with different immune-reactivity revealed the presence of viral proteins in two different areas of the cells presumably implicated in different phases of the viral replication. Furthermore, also in olive flounder has been revealed the participation of macrophages in this viral infection and a presumptive role of the spleen in limiting the spreading of LCDV in the tissues.