The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants’ physical defence barriers.