Aceh patchouli (Pogostemon cablin Benth.) is an aromatic plant that produces essential oil, with patchouli alcohol (PA) as the primary compound determining its quality. This study aimed to investigate the effects of delignification using the fungus Trichoderma viride on PA content, other chemical components in patchouli oil, and the functional group characteristics of the oil. A factorial experimental design was employed, with the first factor being leaf delignification (control without delignification, natural delignification, and delignification by T. viride for 5 days), and the second factor being the extraction method (hydrodistillation and steam distillation). PA content and the composition of volatile compounds were analyzed using Gas Chromatography–Mass Spectrometry (GC–MS), while Fourier-Transform Infrared (FTIR) spectroscopy was used to characterize functional groups. This study provides a novel integrated approach by combining biological delignification using T. viride with different distillation techniques and spectroscopic–multivariate analysis to systematically evaluate changes in patchouli oil composition and extraction efficiency. The results indicated that delignification by T. viride significantly increased PA content, with the highest concentration reaching 61.12%, compared to 37.96% in the control treatment. The levels of other compounds, such as caryophyllene, decreased following delignification, indicating a shift in chemical composition. Delignification treatment had a significant effect on PA content, whereas the distillation method showed no significant difference. FTIR analysis revealed consistent major functional groups, including O–H and C–H vibrations, suggesting that the primary chemical functionalities of the oil were retained. However, these results should be interpreted cautiously due to the complex mixture nature of essential oils and the overlapping of spectral bands. Overall, T. viride demonstrated potential as an effective and environmentally friendly biological pretreatment agent to enhance patchouli oil quality by improving PA yield without evident alteration of its dominant functional groups.