Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson's disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD.