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. Here, VIM is linked to breast cancer.