Apple scab disease caused by the fungus Venturia inaequalis is a devastating disease that seriously affects quality and yield of apples. In order to understand the mechanisms involved in scab resistance, we performed gas chromatography-mass spectrometry based metabolomics analysis of the cell culture of scab resistant cultivar 'Florina' and scab susceptible cultivar 'Vista Bella' both prior -to and -following treatment with V. inaequalis elicitor (VIE). A total 21 metabolites were identified to be altered significantly in 'Florina' cell cultures upon VIE-treatment. Among 21 metabolites, formation of three new specialized metabolites aucuparin, noraucuparin and eriobofuran were observed only in resistant cultivar 'Florina' after the elicitor treatment. The score plots of principal component analysis (PCA) exhibited clear discrimination between untreated and VIE-treated samples. The alteration in metabolite levels correlated well with the changes in the transcript levels of selected secondary metabolite biosynthesis genes. Aucuparin, noraucuparin and eriobofuran isolated from the 'Florina' cultures showed significant inhibitory effect on the conidial germination of V. inaequalis. The results expand our understanding of the metabolic basis of scab-resistance in apple and therefore are of interest in apple breeding programs to fortify scab resistance potential of commercially grown apple cultivars.