AbstractCyprinid herpesvirus 3 (CyHV‐3), also known as koi herpesvirus (KHV), is an aetiological agent of a virulent and lethal disease in common and koi carp. In this study, we examined in vitro the anti‐CyHV‐3 activity of acyclovir (ACV), nucleoside analogue commonly used against human herpesviruses, as well as acyclovir monophospate (ACV‐MP). The cytotoxicity of theACVand theACV‐MPfor two common carp cell lines,CCB(Common carp brain) andKF1 (Koi carp fin 1), was determined by means ofMTTand crystal violet assays. In subsequent studies, the concentration of 66.67 μM was applied. TheACVand theACV‐MP(66.67 μM) inhibited a cytopathic effect (CPE) induced by the CyHV‐3 virus in theCCB(ACVby 66%,ACV‐MPby 58%) and theKF1 (ACVby 25%,ACV‐MPby 37%). The viral load measured by the means of TaqManqPCRwas reduced in a range of 67%–93% depending on the analogue, the cell line and the time of incubation. The expression of viral genes (ORF149,ORF3,ORF134 andORF78) inCCBcells infected with the CyHV‐3 was strongly downregulated within the range of 78%–91%. In summary, both theACVand theACV‐MPcan inhibit CyHV‐3 replication in vitro.