Polyploidization in plants, which involves doubling or further multiplying of genome, has the potential to improve the constituents that make medicinal plants, like Ajuga reptans, attractive to the pharmaceutical, cosmetics, and food production industries; botanical pesticide effects could also be derived. The aim of this study was to determine how artificial polyploidization in A. reptans plants affected the composition and quantity of biologically active substances from the glycoside and phytoecdysone families. Diploids and artificial tetraploids of A. reptans were analyzed. Changes in the contents of trans-teupolioside, trans-verbascoside, and 20-hydroxyecdysone were evident in the aboveground parts of the cultivated plants (e.g., leaves and flowers). The tetraploid lines of Ajuga plants displayed variability in, and increased levels of, trans-teupolioside and trans-verbascoside content. The 20-hydroxecdysone content was slightly higher in tetraploids. These findings indicated that Ajuga tetraploids could be used in breeding programs to enhance the yield of substances with potential medicinal and industrial applications.