Display omitted •TLC and HPLC–QTOF/MS techniques were employed to compare the chemical profiles of CWT and CML.•A HPLC–DAD method was developed for quantification of eight major compounds in CWT and CML.•The antioxidant activity of CWT and CML were compared by DPPH, FRAP and ABTS assay.•The results indicated that there were significant chemical differences between CWT and CML, and CWT had higher antioxidant activity than CML. Citri Fructus (CF), the mature fruit of Citrus wilsonii Tanaka (CWT) or Citrus medica L. (CML), is an important citrus by-product with health promoting and nutritive properties. The present study compares the chemical and biological differences of CWT and CML. Thin layer chromatography and high performance liquid chromatography, coupled with quadrupole time-of-flight tandem mass spectrometry techniques, were employed to compare the chemical profiles of CWT and CML. A total of 25 compounds were identified and the results indicated that there were significant differences in chemical composition between the two CF species. The quantitative results obtained by HPLC coupled with diode array detector method demonstrated that naringin was present in the highest amounts in CWT, whilst nomilin was the most dominant constituent in CML. It was also found that CWT had significantly higher free radical-scavenging activity than CML.