To investigate the effects of antibiotics and disinfectants—commonly used aquaculture agents—on fish skin immune responses and mucosal microbiota, zebrafish were exposed to 25 μg/mL rifampicin (RIF), 10 μg/mL oxytetracycline (OTC), 2 mg/L potassium permanganate (KMnO4), and 0.1 mg/L chlorine dioxide (ClO2) for 12 hours. Skin immune responses and microbial community dynamics were analyzed via quantitative real-time PCR (qPCR) and 16S rRNA high-throughput sequencing. Results demonstrated that exposure to antibiotics and disinfectants significantly upregulated the expression of inflammation-related genes in zebrafish skin, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), compared to the control group (P<0.05). Immunoglobulin IgM, complement component C3, and mucin Muc2 were also markedly elevated post-exposure (P<0.05). Furthermore, antibiotic and disinfectant treatments significantly reduced skin microbiota diversity and richness, accompanied by structural shifts in microbial communities. Alpha-diversity indices (Chao1, Shannon, Ace, and Sobs) all showed significant decreases (P<0.05). Principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) of microbial profiles across five groups revealed clear separation between control and treatment clusters. Taxonomic analysis indicated that at the phylum level, the relative abundance of Proteobacteria increased significantly, whereas Bacteroidota and Firmicutes declined. At the genus level, Acinetobacter exhibited substantial enrichment in all treatment groups, particularly reaching 65.36% in the KMnO4-treated group. These findings suggest that antibiotics and disinfectants disrupt skin microecological balance through dual mechanisms: (1) excessive activation of inflammatory pathways, leading to immune homeostasis imbalance; and (2) proliferation of opportunistic pathogens, resulting in microbiota dysbiosis and compromised microbial barrier function.