Ostracod assemblages from Lake Trasimeno (Umbria, central Italy), the largest endorheic lake in Italy, were investigated relating their species distribution and ecology to modern physical, chemical and biological parameters. Nineteen living species were collected in the lake (Darwinula stevensoni, Candona (Candona) candida, Candona (Neglecandona) angulata, Fabaeformiscandona fabaeformis, Pseudocandona marchica, Cypria ophtalmica, Ilyocypris gibba, I. salebrosa, I. getica, Cypridopsis vidua, Eucypris virens, Trajancypris clavata, Herpetocypris helenae, Heterocypris salina, H. incongruens, Isocypris beauchampi, Cyprideis torosa, Limnocythere inopinata, and L. stationis). All the identified species belong to the fresh-water Italian ostracod fauna but Cyprideis torosa is documented in an oligohaline athalassic lacustrine environment in Italy for the first time. The occurrence of Ilyocypris salebrosa represents the southernmost record in Italy and the westernmost in Eurasia. The recovery of Limnocythere stationis represents the westernmost record in Eurasia. The distribution of the different ostracods recovered in Lake Trasimeno is linked to the dominant physical and chemical parameters for each ecological niche. Physical and chemical data along with substratum type, grain-size and presence of aquatic macrophytes have been related to different ostracods using a multivariate analyses approach (NMDS, CCA, Spearman’s rank correlation test). These results allow to differentiate several ecological niches within the lake and indicate that the main parameters affecting the ostracod assemblages are the aquatic macrophyte coverage, the Total Organic Carbon (TOC) and, to a lesser extent, temperature and type of substrate. Cyprideis torosa and Candona (Neglecandona) angulata have been recovered both in the distal part of the lake and in the lakeshore area. In both cases they are associated with scarce or absent aquatic macrophytes and low amounts of TOC. The alternate dominance of these two species in the distal deeper assemblages seems to be mainly linked with the bottom oxygen availability, being C.(N.) angulata dominant in the most oxygen-depleted sediments and C. torosa dominant in higher oxygen conditions. Along the lakeshore area they are often discovered together with other prevailing species, such as Cypridopsis vidua that is common in very shallow to shallow (20-140 cm) sites with high TOC content, abundant macrophytes and algae, and Limnocythere inopinata, which dominates slightly deeper areas (around 150-210 cm) where the previous species are almost absent. The Spearman’s rank correlation test showed significant positive correlation between some ostracods and macrophyte species.