This study aimed to develop an efficient micropropagation protocol for Plectranthus amboinicus (Lour.) Spreng, evaluate the genetic fidelity of in vitro-regenerated plants using RAPD molecular markers, and perform a biochemical assessment of micropropagated material compared with acclimatized plants. For the initiation of in vitro cultures, explants (uninodal fragments) were inoculated on Murashige and Skoog (MS) medium without plant growth regulators (PGRs). Various plant growth regulator treatments were tested to evaluate the in vitro regenerative potential of the explants. The highest multiplication rate (3.43 ± 0.21 shoots per explant) was obtained on MS basal medium supplemented with 3 mg/L 6-benzylaminopurine (BAP) and 3 mg/L gibberellic acid (GA₃), while the greatest shoot length (3.22 ± 0.22 cm) was achieved on MS medium containing 1 mg/L BAP and 3 mg/L GA₃. Indole-3-butyric acid (IBA) (0.5 mg/L), applied alone or in combination with GA₃ (3 mg/L), was the most effective treatment for inducing a well-developed root system, facilitating successful acclimatization of regenerated plants (100% survival rate). In vivo rooting of shoots simultaneously with acclimatization was also tested. The best results (100% rooting rate) were obtained using perlite or Jiffy peat pellets as the rooting substrate, and the shoots were treated with Radi Stim. Molecular analyses confirmed the genetic fidelity of the micropropagated plants. Biochemical analyses revealed higher levels of phenolic compounds and flavonoids in in vitro-cultured plants than in acclimatized plants. These results validate the micropropagation protocol morphologically, genetically, and biochemically, and highlight in vitro culture as an efficient approach for producing high-quality P. amboinicus plant material and for the sustainable production of valuable bioactive compounds.