Zanthoxylum zanthoxyloides is an endangered African tree producing numerous bioactive substances including antileukemic and antisickling agents. Here, the potential of Z. zanthoxyloides hairy root cultures was tested for the production of bioactive substances with limited natural resources. The efficiency of Agrobacterium rhizogenes LBA9402-mediated transformation of leaf material was evaluated using different techniques. An optimal transformation frequency of 77% was obtained after 11 days by inoculating A. rhizogenes directly onto the central vein of 14-week-old leaves followed by a co-cultivation period of 3 days. Different treatments in immersion mode (manual wounding, acetosyringone, CaCl2, ultrasonication) never exceeded these results. A maximum growth rate of 0.37 cm/day was determined during the exponential phase. Liquid chromatography-diode array detection analysis showed the presence of skimmiamine, sesamine, chelerythrine, and chelerythrine derivatives in Z. zanthoxyloides hairy root lines. The maximum production of skimmiamine and chelerythrine in 28-day-old hairy root cultures was 45 ± 2 and 107 ± 4 mg/100 g dry weight, respectively. The present results highlight the potential of Z. zanthoxyloides hairy root cultures for the sustainable production of skimmiamine and chelerythrine.