Traditional Chinese Medicine (TCM) has accumulated valuable experience from ancient China while mixed with impurities. Plants from the Rubiaceae family are widely distributed in China, with a long history of use and a significant amount of related literature on Traditional Chinese Medicine. This study used ethanol extracts from three Rubiaceae plants, namely Galium spurium L., Rubia cordifolia L., and Hedyotis corymbosa L., to conduct antimicrobial experiments against Escherichia coli and Bacillus subtilis. Among them, G. spurium and R. cordifolia were confirmed to have antimicrobial capabilities, while H. corymbosa did not show antimicrobial ability. In the E. coli experiment, the diameter of the inhibition zone of G. spurium extract was approximately 1.48 cm, and that of R. cordifolia extract was about 1.03 cm. For B. subtilis, the inhibition zone diameter of G. spurium extract was approximately 1.49 cm, and that of R. cordifolia extract was about 1.48 cm. This experiment, conducted under modern research methods, confirmed the antimicrobial abilities of these three plants.