Background <p>In vitro rooting remains a challenge for many woody species, including <i>Quercus robur</i>. While auxins combined with activated charcoal (AC) have traditionally been used, <i>Q. robur</i> explants exhibit a strong dependence on cytokinins (CKs). This study introduces a novel approach combining AC with either 6-benzylaminopurine (BAP) or meta-topolin (mT) during the rooting phase to evaluate morphophysiological and hormonal responses.</p> Methods <p>Eight treatments were tested: 0 µM CK + 2&#xa0;g L⁻¹ AC; 2, 4, and 8 µM BAP + 2&#xa0;g L⁻¹ AC; 2, 4, and 8 µM mT + 2&#xa0;g L⁻¹ AC; and to isolate the effect of AC, a treatment without exogenous CK and AC (0 µM CK + 0&#xa0;g L⁻¹ AC) was also included. Morphophysiological and hormonal analyses were performed after 40 days of cultivation.</p> Results <p>Plantlets grown without AC exhibited a high shoot-to-root biomass ratio, indicating reduced growth balance. Treatments with 0–8 µM BAP combined with 2&#xa0;g L⁻¹ AC reduced the ratio of vascular cylinder area to total root cross-section by 16.5% compared to the other treatments. Notably, mT-treated plantlets developed root systems with improved anatomical features conducive to transport efficiency. CKs at 8 µM significantly enhanced stem thickening via stimulated cambial activity. Additionally, CK + AC treatments increased chlorophyll (Chl <i>a</i> + <i>b</i>) and carotenoid (Car) contents, while the absence of both CK and AC resulted in higher Car/Chl <i>a</i> + <i>b</i> ratios and an increased photosystem II closure rate (M₀), indicating stress. Hormonally, CK-free treatments led to elevated indole-3-acetic acid (IAA) and reduced jasmonic acid (JA) levels, suggesting an imbalance.</p> Conclusion <p>Overall, the combination of AC and 4–8 µM mT during rooting enhanced secondary growth and improved the hormonal balance and morphophysiological traits of <i>Q. robur</i> plantlets.</p>