This study reports the mosquito collections conducted in July-August 2013 in Castiglione del Lago (Umbria Region, Italy) during the entomological surveillance within the West Nile national control program. The mosquito collections showed the noteworthy occurrence of Anopheles hyrcanus s.l. (n = 156; 35.8% of the whole sample), a relatively rare mosquito species in Italy, with molecular analyses con rming that these mosquitoes belong to An. hyrcanus s.s. The same catching site was characterized by a relevant richness in mosquito species, in particular the following were found: Anopheles maculipennis s.l. (n = 146; 33.5% composed by 80% Anopheles melanoon, 13.3% Anopheles maculipennis s.s. and 6.7% Anopheles atroparvus), Culex pipiens s.l. (n = 116; 26.6% composed by 90% Cx. p. pipiens, 5% Cx. p. molestus and 5% Cx. p. pipiens/molestus), Culiseta annulata (n = 10; 2.3%), Aedes albopictus (n = 3; 0.7%), and Anopheles claviger (n = 5; 100%).