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Integrative taxonomic studies of macrofungal diversity in the Brijuni National Park (Istria County, Croatia) led to the discovery of a second species of <i>Inocybe</i> (Agaricales, Inocybaceae) new to science. <i>Inocybe istriaca</i> sp. nov. is described on the basis of morphological, ecological, and multigene phylogenetic analyses, and its placement within the family Inocybaceae is discussed. The combination of most important morphological characters that distinguish <i>I. istriaca</i> from the other similar <i>Inocybe</i> species are smooth, (sub)amygdaliform, (sub)phaseoliform, or ellipsoid basidiospores (ca. 8.5–12 × 5–7 μm), large basidia (36–45 × 9–15 μm), mostly (sub)fusiform and weakly thick-walled (up to 1.5 μm) metuloid pleurocystidia, and lamellar edge and stipe apex partially covered by a dark resinous substance. The species was collected on the edge of grassland and Mediterranean evergreen holm oak (<i>Quercus ilex</i>) forest. In this study, a total of 14 DNA sequences from four <i>Inocybe</i> species were generated. Two-gene (ITS, LSU) and four-gene (ITS, LSU, <i>rpb2</i>, <i>tef1</i>) phylogenetic analyses confirmed the status of <i>I. istriaca</i> as an independent species.