Studies in both humans and animal models demonstrated that chronic alcohol/e-cigarette (e-Cig) exposure affects mitochondrial function and impairs barrier function in brain microvascular endothelial cells (BMVECs). Identification of the signaling pathways by which chronic alcohol/e-Cig exposure induces mitochondrial damage in BMVEC is vital for protection of the blood–brain barrier (BBB). To address the issue, we treated human BMVEC [hBMVECs (D3 cell-line)] with ethanol (ETH) [100 mM], acetaldehyde (ALD) [100 μM], or e-cigarette (e-Cig) [35 ng/mL of 1.8% or 0% nicotine] conditioned medium and showed reduced mitochondrial oxidative phosphorylation (OXPHOS) measured by a Seahorse analyzer. Seahorse data were further complemented with the expression of mitochondrial OXPHOS proteins detected by Western blots. We also observed cytosolic escape of ATP and its extracellular release due to the disruption of mitochondrial membrane potential caused by ETH, ALD, or 1.8% e-Cig exposure. Moreover ETH, ALD, or 1.8% e-Cig treatment resulted in elevated purinergic P2X7r and TRPV1 channel gene expression, measured using qPCR. We also demonstrated the protective role of P2X7r antagonist A804598 (10 μM) in restoring mitochondrial oxidative phosphorylation levels and preventing extracellular ATP release. In a BBB functional assay using trans-endothelial electrical resistance, we showed that blocking the P2X7r channel enhanced barrier function. In summary, we identified the potential common pathways of mitochondrial injury caused by ETH, ALD, and 1.8% e-Cig which allow new protective interventions. We are further investigating the potential link between P2X7 regulatory pathways and mitochondrial health.