Parkinson’s disease (PD) requires multi-target therapies to mitigate the toxicity of α-synuclein (α-Syn) aggregation in the brain. Tetracyclines, particularly doxycycline, have demonstrated multimodal neuroprotective effects, both in vitro and in vivo. The non-antibiotic derivative of doxycycline 4- dedimethylamino-12a-deoxydoxycycline (DDOX), has been recently shown to rescue neurons from oxidative injury. Here, we demonstrate that DDOX showcases a diverse range of mechanisms targeting α-Syn aggregates. Notably, DDOX inhibited the aggregation of αSyn and the seeding ability of α-Syn pre-formed fibrils (PFF) in biophysical and cellular assays. In addition, the compound ameliorated total and phospho-α-Syn relocalization, triggered by exogenous α-Syn PFF. Surprisingly, DDOX drastically mitigated lysosomal stress induced by these aggregates. Moreover, we determined that DDOX effectively impeded the internalization of fluorescently labeled α-Syn PFF. Biophysical techniques and molecular docking simulations suggest that DDOX binds to hydrophobic patches on α-Syn fibrils. Our findings reveal novel neuroprotective attributes of tetracyclines, wherein a direct extracellular interaction between DDOX and α-Syn aggregated species mitigates their intracellular impact. These results provide a promising foundation for DDOX, a drug that aims at interfering with the intracellular seeding, propagation and uptake of α-Syn fibrils in neurodegenerative conditions.