The present study considered the in vitro propagation of Brunfelsia pauciflora utilizing various cytokinins, as well the estimation of the antioxidant, total phenolics (TPs), and flavonoids (TFs) for shootlets of each treatment. The explants were sterilized with 0.2% mercuric chloride (20 min) and then cultured on Murashige and Skoog medium (MS) supplemented with three cytokinins; benzyl aminopurine (BAP), 6-Furfuryl-aminopurine (Kin), and isopentenyl adenine (2iP) at different concentrations. The highest shootlets number and fresh weight were obtained using 1.0 mg/L BAP, while shootlets length is increased using 2.0 mg/L kin. Additionally, significant numbers of leaves were observed for the plantlets supplemented with kin (1.0 & 3.0 mg/L) and 2iP (2.0 & 3.0 mg/L). The shootlets were rooted one month later with different strengths (full, ¾, ½, or ¼) on MS media without or with 1.0g/L activated charcoal (AC). Shootlet cultured on full strength MS medium with AC gave the longest root, while the greatest numbers of roots were recorded using ¾ strength without AC. All the rooted shootlets were acclimatized on peat moss and recorded with the longest root length and greatest leaves and roots numbers. Alternatively, the methanolic extract of shootlets supplemented with 2iP (3.0 mg/L) yielded the maximum antioxidant effect (89.89 ± 0.28 mg Trolox /g extract), TPs (57.574±0.82 mg Gallic/g extract), and TFs (46.146 ± 0.31 mg Catechin/g extract) compared with the control (MS free) and mother plant. In conclusion, the antioxidant potential was shown to be strongly correlated with the TFs and the number of leaves /shootlet.