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Fungal infections are a global public health challenge, especially among immunocompromised patients. <i>Basidiomycetous</i> yeasts, such as <i>Rhodotorula mucilaginosa</i>, have emerged as opportunistic pathogens, but have received less attention than <i>Cryptococcus neoformans</i>. This study aimed to characterize the polysaccharides of <i>R. mucilaginosa</i> and compare them with those of <i>C. neoformans</i>, analyzing their clinical implications. Comprehensive physicochemical, mechanical, and ultrastructural analyses of polysaccharides from both species were performed, revealing correlations with virulence and pathogenicity. <i>R. mucilaginosa</i> cells are surrounded by a capsule smaller than that produced by <i>C. neoformans</i>, but with similar polysaccharides. Those polysaccharides are also secreted by <i>R. mucilaginosa</i>. Cross-reactivity with <i>R. mucilaginosa</i> was observed in a diagnostic <i>C. neoformans</i> antigen test, using both <i>in vitro</i> and <i>in vivo</i> samples, highlighting the need for more reliable tests. Some <i>R. mucilaginosa</i> strains exhibited virulence comparable to that of <i>C. neoformans</i> in an invertebrate experimental model (<i>Tenebrio molitor</i>). This study contributes to a deeper understanding of yeast pathogenicity and virulence, highlighting the need for more accurate diagnostic tests to improve the differential diagnosis of infections caused by basidiomycetous yeasts.