The present study aimed to investigate the effect of Pseudomonas aeruginosa (PA) lipopolysaccharide (LPS) on Candida albicans biofilm formation in a secretory otitis media (SOM) rat model and its potential impact on the integrity of the blood-labyrinth barrier. LPS was extracted from PA standard bacteria, and the effect on the biofilm formation of Candida albicans was analyzed. A total of 20 Sprague-Dawley rats were randomly divided into control group (35 μL saline) and SOM group (35 μL PA LPS), with 10 rats in each group. The threshold of auditory brainstem response was measured. The extravasation rate of blood vessels was detected by intravenous injection of Albumin-FITC. Levels of interleukin (IL)-4, IL-6, IL-17, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β in serum were detected. The residual amount of Albumin-FITC and the expression of ZO-1, Occludin, and VE-cadherin were detected in stria vascularis tissues. PA LPS inhibited the biofilm formation of Candida albicansin vitro. Compared to control group, the SOM group had a markedly increased brainstem response threshold and lymphedema in the middle ear mucosa (P≤0.05), the serum IL-4 in SOM group decreased (P≤0.05), and the levels of IL-6, IL-17, TNF-α, TGF-β, and IFN-γ increased (P≤0.05). As compared to the control group, the SOM group had an obvious raise in the extravasation rate of vascular Albumin-FITC (P≤0.05), and residual amount of Albumin-FITC in the stria vascularis (P≤0.05), and clear reductions in the mRNA expression of ZO-1 and Occludin and the protein expression of ZO-1, Occludin, and VE-cadherin in the stria vascularis (P≤0.05). PA LPS can inhibit the formation of Candida albicans biofilm in vitro, PA LPS-induced acute SOM directly disrupts the integrity of the blood-labyrinth barrier by increasing vascular permeability and reducing the expression of barrier proteins such as ZO-1 and Occludin.