| Summary: | Herbal and traditional medicines can play a pivotal role in combating cancer and neglected tropical diseases. <i>Ajuga bracteosa</i>, family Lamiaceae, is an important medicinal plant. The genetic transformation of <i>A. bracteosa</i> with <i>rol</i> genes of <i>Agrobacterium rhizogenes</i> further enhances its metabolic content. This study aimed at undertaking the molecular, phytochemical, and in vitro biological analysis of <i>A. bracteosa</i> extracts. We transformed the <i>A. bracteosa</i> plant with <i>rol</i> genes and raised the regenerants from the hairy roots. Transgenic integration and expression of <i>rolB</i> were confirmed by conventional polymerase chain reaction (PCR) and qPCR analysis. The methanol: chloroform crude extracts of wild-type plants and transgenic regenerants were screened for in vitro antibacterial, antihemolytic, cytotoxic, anticancer, and leishmanial activity. Among all plants, transgenic line 3 (ABRL3) showed the highest expression of the <i>rolB</i> gene. Fourier transform infra-red (FTIR) analysis confirmed the enhanced number of functional groups of active compounds in all transgenic lines. Moreover, ABRL3 exhibited the highest antibacterial activity, minimum hemolytic activity (CC<sub>50</sub> = 7293.05 ± 7 μg/mL) and maximum antileishmanial activity (IC<sub>50</sub> of 56.16 ± 2 μg/mL). ABRL1 demonstrated the most prominent brine shrimp cytotoxicity (LD<sub>50</sub>39.6 ± 4 μg/mL). ABRL3 was most effective against various human cancer cell lines with an IC<sub>50</sub> of 57.1 ± 2.2 μg/mL, 46.2 ± 1.1 μg/mL, 72.4 ± 1.3 μg/mL, 73.3 ± 2.1 μg/mL, 98.7 ± 1.6 μg/mL, and 97.1 ± 2.5 μg/mL against HepG2, LM3, A549, HT29, MCF-7, and MDA-MB-231, respectively. Overall, these transgenic extracts may offer a cheaper therapeutic source than the more expensive synthetic drugs.
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