Puccinia triticina (Pt) is an important pathogen of wheat. While breeding programmes develop resistant wheat cultivars to mitigate the effects of such rust-causing pathogens, the emergence of new rust races with wider virulence mandates the implementation of other control strategies. Our study investigated whether acidic pH conditions affected selected Carbohydrate-Active Enzymes (CAZymes) in Pt-inoculated Thatcher + Lr9 (IR) wheat compared to those found in the Thatcher (IS) wheat. The β-glucosidase and amyloglucosidase activity levels significantly increased in IR compared to the control from 1 to 14 days post-inoculation (dpi). In contrast, activity levels of invertase did not change in the IR wheat relative to the control at 1 and 7 dpi, but were significantly reduced in the IR plants at 14 dpi. The IS had higher activity of all three hexose-producing enzymes under acidic conditions. These enzyme activities could be increased in the IS to produce hexose sugars required by Pt to develop and advance infection. The phenotypic analysis supported this view because leaf rust disease symptoms were only visible in the IS plants. For cell wall loosening-related enzymes, the IR displayed higher activity of exoglucanase, xylanase and peroxidase enzymes compared to IS. The liquid chromatography–mass spectrometry analysis showed IR had higher concentrations of complex oligosaccharides compared to the IS. Thus, we concluded that the higher exoglucanase, xylanase and peroxidase activity could be involved in cell wall loosening under acidic conditions, while oligosaccharides could be building-blocks for synthesizing cell wall barriers that apprehend Pt growth in inoculated Thatcher + Lr9.