Europe was officially declared free from malaria in 1975; nevertheless, this disease remains a potential problem related to the presence of former vectors, belonging to the Anopheles maculipennis complex. Autochthonous-introduced malaria cases, recently reported in European countries, together with the predicted climatic and environmental changes, have increased the concern of health authorities over the possible resurgence of this disease in the Mediterranean Basin. In Italy, to study the distribution and bionomics of indigenous anopheline populations and to assess environmental parameters that could influence their dynamics, an entomological study was carried out in 2005-2006 in an at-risk study area. This model area is represented by the geographical region named the Maremma, a Tyrrhenian costal plain in Central Italy, where malaria was hyperendemic up to the 1950s. Fortnightly, entomological surveys (April-October) were carried out in four selected sites with different ecological features. Morphological and molecular characterization, blood meal identification, and parity rate assessment of the anophelines were performed. In total, 8274 mosquitoes were collected, 7691 of which were anophelines. Six Anopheles species were recorded, the most abundant of which were Anopheles labranchiae and An. maculipennis s.s. An. labranchiae is predominant in the coastal plain, where it is present in scattered foci. However, this species exhibits a wider than expected range: in fact it has been recorded, for the first time, inland where An. maculipennis s.s. is the most abundant species. Both species fed on a wide range of animal hosts, also showing a marked aggressiveness on humans, when available. Our findings demonstrated the high receptivity of the Maremma area, where the former malaria vector, An. labranchiae, occurs at different densities related to the kind of environment, climatic parameters, and anthropic activities.