Microbial spoilage poses a significant challenge to fresh-cut apples during storage, impacting quality and consumer health. This study explores pyridoxine’s (vitamin B6; VB6) potential mode of action to mitigate microbial spoilage in fresh-cut apples through physiological, transcriptomic, and metagenomic analysis. VB6 notably reduces microbial spoilage at room temperature by suppressing bacterial genera (Pseudomonas, Curtobacterium, Frigoribacterium) and fungal genera (Alternaria, Penicillium, Aureobasidium, Pseudogymnoascus) proliferation. Spoilage-causing identified biomarkers include species related to Pseudomonas, Curtobacterium, Frigoribacterium, and Penicillium. KEGG analysis suggests correlations of induced disease resistance with tropane alkaloids biosynthesis, plant-pathogen interaction, and phenylpropanoid biosynthesis pathways in fresh-cut apples. Moreover, VB6 treatment enhanced the antioxidant system via maintaining higher total phenolic contents, total flavonoid contents, and ascorbic acid under elevated catalase, superoxide dismutase, and ascorbate peroxidase enzymes activities, induced disease resistance under higher enzymes activities including phenylalanine ammonialyase, chitinase, cinnamate 4-hydroxylase, β-1,3-glucanase, and 4-coumarate: co-enzyme A ligase enzymes activities, maintained elevated VB6 contents, and reduced H2O2 accumulation. Notably, 6 elevated transcription factors involving WRKY, NAC and MYB might regulate disease resistance pathways in fresh-cut apples under VB6 treatment. This research establishes a theoretical and practical foundation for preserving post-processing quality of fresh-cut apples by restraining microbial proliferation through the proposed VB6-induced disease resistance regulatory mechanism.