Background: Ocimum sanctum (L.) is a well-known extensively employed Indian medicinal shrub in traditional and modern therapeutics. Hairy roots exhibit similar or superior capability to inhibit oxidants responsible for many disorders. Objectives: Agrobacterium-mediated transformation protocol has developed for O. sanctum which influences T-DNA delivery. The antioxidant, antiacetylcholinesterase (AChE) potential, and in vivo study of hairy root extract of O. sanctum has been evaluated. Materials and Methods: Sterilized O. sanctum shoot tips had grown in vitro by employing Murashige and Skoog basal media, Benzyl amino purine (BAP) (0.5 mg/L), and other additives. In vitro grown leaf explants were infected by Agrobacterium rhizogenes ATCC 15834 for various time durations. Polymerase chain reaction (PCR) employed to confirm the genetic assimilation of Agrobacterium. The hairy root extract's antioxidant potential has been tested by diphenyl picric hydroxyl (DPPH) and ferrous ion reducing assay (ferrous reduced antioxidant power [FRAP]) along with anti-AChE potential. In vivo study was done employing behavioral and biochemical estimation. Results: For in vitro culturing of O. sanctum, administration of BAP (0.5 mg/L) shows growth rate of 32.7% ±0.19% in 7 days. The Agrobacterium-infected explants show the good yield of hairy root in 1 h of infection duration. The PCR results show the genomic integration of transgene rol A and rol C. The methanolic extracts of hairy roots showed potential antioxidant activities for DPPH-free radical (66.15% ±0.34%) and FRAP assay (226.35 ± 0.17TE/gDW of the sample). These extracts effectively inhibit AChE (28.710 ± 0.26 μg/mL at 3 min incubation duration). In in vivo study, the pretreatment of OSM (200 and 400 mg/kg) exhibited significant increase of spatial and long term after administration of scopolamine. Conclusion: The obtained results exhibited a simple protocol for micropropagation and transgenic roots production with competently targeting AChE and related disorders.