Introduction: The trade in marine ornamental fish is a multi-billion-dollar industry involving over 50 exporting and importing countries. Europe is one of the three major importers of marine ornamental fish, along with the United States and Japan. Megalocytiviruses are associated with severe mortality in ornamental fish aquaculture, including both freshwater and marine species. The international trade in live ornamental fish has aided the spread of these viruses. So far, megalocytiviruses have been detected in few European countries (Belgium and Germany), but, to the best of our knowledge, they have never been detected in Italy. Methodology: In the context of a health surveillance programme on imported ornamental fish, histological lesions consistent with megalocytivirus infection were pointed out in two fish dead during the quarantine period. Case 1# referred to one neon damselfish (Pomacentrus coelestis) out of 10 subjects involved in a 100% mortality event in June 2018, whereas Case 2# was a prickly leatherjacket (Chaetodermis penicilligerus) dead after showing lethargic behaviour in June 2021. To investigate the presence of the megalocytivirus genome, FFPE tissue samples, including the areas with enlarged cells, were subjected to DNA extraction and PCR/real-time PCR analysis using two protocols previously described. PCR products were sequenced and subjected to phylogenetic analysis. Results: The histological investigation showed in both species the presence in the kidney, spleen, liver, heart, gills, intestine of multiple hypertrophic cells containing granular to smudgy basophilic intracytoplasmic inclusions. Often, these cells were clearly endothelial cells. The presence of megalocytivirus DNA was pointed out in the two fish samples resulting positive to the real-time PCR and being identified as megalocytiviruses. Phylogenetic analysis of a fragment of the MCP gene from Case 2# showed its clustering within the RSIV-like group. Conclusions: The presence of megalocytivirus was pointed out in imported marine ornamental fish for the first time in Italy. The viral DNA was detected in Pomacentrus coelestis and Chaetodermis penicilligerus, two marine ornamental species that, so far, have never been associated with megalocytivirus infection. Due to the high risk of importing megalocytiviruses through the ornamental fish trade, a strengthening of the surveillance programmes and quarantine measures is recommended.