Aloe vera is a perennial, succulent plant which has been used in folk medicine for wound-healing, as its roots have been reported to exhibit remarkable antimicrobial properties. However, collecting the roots is detrimental to plant growth and development. Hence, an alternative system of producing A. vera roots must be developed. Tissue culture is a potential tool to obtain A. vera root biomass, which can serve as raw materials for the large-scale production of antimicrobial compounds. In this study, individual shoots taken from the multiple shoot culture of A. vera were induced to form roots in MS medium supplemented with 1 μM of naphthaleneacetic acid (NAA), indolebutyric acid (IBA), and indoleacetic acid (IAA). Among these 3 auxins tested, NAA was found to be the most effective auxin for root induction in tissue culture of A. vera, resulting in the induction of 10.7 ± 4.0 roots. IBA and IAA induced a lower number of roots at 2.0 ± 1.0 and 1.7 ± 1.2, respectively. Two-month-old in vitro culture-derived roots were extracted and assayed for antimicrobial activity. The extract from in vitro culture-derived roots was found to have remarkable antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria Escherichia coli and Ralstonia solanacearum, and the molds Fusarium oxysporum and Cercospora capsici with minimum inhibitory concentration (MIC) values of 15.60, 31.25, 62.50, 62.50, 31.25, and 62.50 μg mL^-1, respectively. In addition, the extract from in vitro culture-derived roots was found to exhibit higher antimicrobial activity than the root extract from field-grown A. vera. This demonstrates the potential of in vitro culture-derived roots of A. vera as a sustainable source of antimicrobial compounds.