Recording the nycthemeral rhythm of sand flies allows the evaluation of the daily activity in different ecotypes, the period of greatest activity, and their degree of anthropophily. We investigated the fauna and the rhythm of sand fly activity in an ecotourism region in Mato Grosso do Sul (MS) state, Brazil. Sand flies were captured monthly, using a Shannon trap for 24 h periods between July 2012 and June 2014. We collected 1,815 sand flies, in which Lutzomyia whitmani (=Nyssomyia whitmani, sensu Galati) and Lutzomyia longipalpis were the most abundant species during the dry season, with activity from 5 p.m.–7 a.m. and 6 p.m.–5 a.m., respectively. Both species require particular attention as vectors of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum in several regions of Brazil, including MS. However, Lutzomyia dispar was more anthropophilic, and was most active between January and March, from 5 p.m. to 5 a.m. Lutzomyia misionensis (=Pintomyia misionensis, sensu Galati) was present throughout both years, active from 4 p.m. to 5 a.m. Other species were active from 5 p.m. to 6 a.m. Due to intense tourism in the months that coincide with a high number of vectors for leishmaniases in Piraputanga, it is essential to determine vector-monitoring strategies in the area by investigating sand fly rhythm while not neglecting other periods of the year when the insects are present.