Leishmaniasis is spreading in Europe, especially in endemic countries such as Italy and Spain, in part due to ongoing climate change and the increase in travel and migration. Although is the main agent responsible for this disease in humans and animals, other species and hybrids have been detected. This highlights the need to continue isolating and characterizing strains from biological samples of infected hosts. In this study, we characterized the recently isolated parasites NAV and TDL, obtained from naturally infected mammals (dogs), and we compared them with the widely distributed and studied strain BCN 150. Both NAV and TDL promastigotes showed a slower growth rate than BCN 150 and were significantly more sensitive to amphotericin B and miltefosine. Furthermore, the expression of the gene (involved in cell cycle and proliferation) was significantly downregulated in NAV and TDL isolates. On the other hand, (implicated in treatment resistance) and (related to the recycling of protein under stress conditions and/or while undergoing a differentiation process and treatment resistance) levels were upregulated, compared to those measured in BCN 150. Both isolates displayed a higher infection capacity (>3 amastigotes per macrophage and >70% of infected macrophages) compared to controls (<2 amastigotes/cells and <50% of infected macrophages). Finally, a higher susceptibility to miltefosine treatment was observed in intracellular NAV and TDL amastigotes. In conclusion, TDL and NAV are novel isolates that might be useful for in vitro and in vivo assays that will allow a better understanding of the parasite biology in Mediterranean areas.