Our key findings include (i) the discovery of Mortalin-derived peptides (#61 and #62) that significantly suppress exNef and EV release in Jurkat T cells and breast cancer cell lines; (ii) demonstration of broad-spectrum EV inhibition by SMRwt and VIMwt peptides across multiple cell types, highlighting their potential as versatile modulators of vesicle trafficking; and (iii) identification of a Vimentin-derived peptide with sequence homology to the Mortalin SMR domain, reinforcing the functional relevance of this conserved motif in mediating protein–protein interactions. This evidence concerns the gene VIM and breast carcinoma.