Patchouli is a plant that produces essential oil. One problem with patchouli is that production remains low due to virus attacks. To address this issue, virus-free seedlings can be produced through tissue culture. This measure requires examining the effectiveness of patchouli regeneration in vitro through histological observations and acclimatization, in as much as effective propagation supports healthy seedlings. The purpose of this study was to determine the optimal propagation for Aceh patchouli via in vitro propagation, through histological observation. It investigated the optimal acclimatization for patchouli to assist patchouli adaptation. The study was carried out in several stages, starting from shoot multiplication, root induction, and acclimatization. Shoot multiplication was carried out on MS medium supplemented with BAP : 0.25 mg/L, 0.50 mg/L, 0.75 mg/L, 1.00 mg/L, 1.25 mg/L, and 1.50 mg/L. Root induction was carried out on MS medium supplemented with NAA : 0 mg/L, 0.2 mg/L, 0.4 mg/L, 0.6 mg/L, and 0.8 mg/L. Finally, the acclimatization was performed on compost under covering treatment. The results demonstrated that the best shoot multiplication, taking place in 6.3 days, was obtained through the 0.25 mg/L BAP treatment. This treatment resulted in the highest shoot proliferation, with a mean of 68.5 shoots per explant, as well as the greatest shoot height (5.1 cm). The best root induction was obtained from NAA 0.2 mg/L, producing a plant length (18.27 cm), plant height (9.60 cm), root length (8.67 cm), and plant fresh weight (1.49 g). During the acclimatization stage, the 12-day covering treatment produced the best seedlings, as evinced by plant length (25.8 cm), shoot height (19.5 cm), root length (7.9 cm), fresh weight (1.85 g), leaf width (3.0 cm), and a survival rate of 100%.