The blood-brain barrier (BBB) preserves CNS homeostasis through selective permeability, preventing neurotoxins and pathogens from entering the brain. Dysbiosis of the gut microbiota has been linked to systemic inflammation and is emerging as a critical factor in neurodegenerative disease pathogenesis, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Metabolites derived from dysbiotic microbiota can disrupt BBB integrity via the gut-brain axis (GBA), triggering neuroinflammation and accelerating neurodegeneration. In this study, we established an in vitro BBB co-culture model by growing human brain microvascular endothelial cells (HuBMECs) with primary human astrocytes on transwell inserts. This model was exposed to disease-relevant stressors, including the primarily pro-inflammatory cytokine interleukin-6 (IL-6), the SARS-CoV-2 spike protein, and cell-free culture supernatant from the Streptococcus pneumoniae strain D39 (D39 SUP). Barrier integrity was evaluated by measuring transendothelial electrical resistance (TEER) and by immunofluorescent detection of the tight junction protein ZO-1, actin, and growth differentiation factor 15 (GDF15). Each stressor caused condition-specific reductions in TEER, consistent with immunofluorescent evidence of disrupted tight junctions and altered actin morphology. Under all pathological treatments, the structural and functional integrity of the BBB was significantly compromised. Additionally, we collected evidence to support an incidental finding: the initiation of an early acute defense mechanism involving both TJP1 and potentially GDF15 expression. These findings highlight early BBB breakdown as a key event in neuroinflammatory pathogenesis and suggest that targeting initial barrier dysfunction could enable novel therapeutic interventions.