Moringa oleifera L. is a fast-growing deciduous tree that is highly nutritious and known to contain essential amino acids, vitamins and various phenolic compounds. The aim of this study was to establish a clean culture using nodal segments of in vitro M. oleifera and to evaluate the plant defence enzymes accumulation of the regenerated plantlets. Surface sterilization of the seeds was done by treating them with 10% or 20% (v/v) of commercial bleach, Clorox® at the duration of 5, 10 or 20 min. Seeds treated with 20% Clorox® for 10 and 15 min produced highest percentage of clean culture and germination rate at 97.5 ± 5.0%. The nodes of the germinated seeds were then used for subsequent multiple shoots induction. The nodes were inoculated onto Murashige and Skoog (MS) media containing either 6-benzylaminopurine (BAP) or thidiazuron (TDZ) at various concentrations (0.5, 1.0 and 2.5 mg/L). All explants in control and BAP (disregard concentrations) produced shoots. The control treatment produced an average of 1.5 ± 0.15 shoots per explant. BAP at the concentration of 0.5 and 1.0 mg/L had significantly increased the average number of shoots per explant to 3.1 ± 0.16 and 3.5 ± 0.51 shoots respectively. On the contrary, all explants on TDZ-enriched media produced calluses instead. Rooting was carried out on MS medium with 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) at various concentrations (0, 0.05, 0.5 and 1.0 mg/L) and combinations. Although media containing 1.0 mg/L IBA gave the highest rooting percentage (36.7%7 ± 12.02) and mean number of roots per shoot (1.05 ± 0.212), there was no statistical difference with other treatments. Media without any growth regulator produced highest mean root length of 2.2 ± 0.322 cm. The assessment of plant defence enzymes activities revealed that phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) had lower activities in in vitro samples compared to soil grown samples. However, peroxidase (PO) activity was higher in in vitro plants. It was speculated that the higher activity of PO was due to the role of the enzyme in rooting of plants in tissue culture. Induced defence enzymes particularly peroxidase could be further explored for their specific antimicrobial natures and use as a natural control agent against phytopathogens.