Thymus pseudopulegioides plantlets were propagated in vitro via direct organogenesis by using Murashige and Skoog (MS) media containing kinetin, thidiazuron, and 6-benzyladenine (BA) individually. Methanol extracts obtained both from plantlets and wild plants were analyzed for their total phenolics and flavonoid contents, then quantified by HPLC. The highest total phenolic (8.83 mg/g as gallic acid equivalent) and total flavonoid (0.92 mg/mL as rutin equivalent) values were from the MS media supplemented with 1.0 mg/L kinetin and 0.5 mg/L BA, respectively. The plantlets grown in those media also showed remarkable antioxidant activities with an IC50 value of 4.77 mu g/mL in DPPH and 100% inhibition in beta-carotene assays, respectively. HPLC analysis proved the production of protocatechuic, caffeic, vanillic, rosmarinic, ferulic, and o-coumaric acids and rutin. Rosmarinic acid production was predominant in natural samples (115.2 mg/100 g dry weight), while the aforesaid phenolic acids were prevalent in plantlets grown on MS media supplemented with KIN or BA at various concentrations. Rutin production was the highest (50.74 mg/100 g dry weight) in the plantlets grown on MS medium containing kinetin (1.0 mg/L). As an economically important chemical, rosmarinic acid was selected as the target chemical and a novel method was introduced to achieve its selective isolation.