This study established the DNA barcoding sequences (matK and rbcL) of three plant species identified in the tribe Clauseneae, namely Clausena excavata, C. harmandiana and Murraya koenigii. The total phenolic and total flavonoid contents, together with the biological activities of the derived essential oils and methanol extracts, were also investigated. Herein, the success of obtaining sequences of these plant using two different barcode genes matK and rbcL were 62.5% and 100%, respectively. Both regions were discriminated by around 700 base pairs and these had resemblance with those of the Clausenae materials earlier deposited in Genbank at a 99–100% degree of identity. Additionally, the use of matK DNA sequences could positively confirm the identity as monophyletic. The highest total phenolic and total flavonoid content values (p < 0.05) were observed in the methanol extract of M. koenigii at 43.50 mg GAE/g extract and 66.13 mg QE/g extract, respectively. Furthermore, anethole was detected as the dominant compound in C. excavata (86.72%) and C. harmandiana (46.09%). Moreover, anethole (26.02%) and caryophyllene (21.15%) were identified as the major phytochemical compounds of M. koenigii. In terms of the biological properties, the M. koenigii methanol extract was found to display the greatest amount of antioxidant activity (DPPH; IC50 95.54 µg/mL, ABTS value 118.12 mg GAE/g extract, FRAP value 48.15 mg GAE/g extract), and also revealed the highest α-glucosidase and antihypertensive inhibitory activities with percent inhibition values of 84.55 and 84.95. Notably, no adverse effects on human peripheral blood mononuclear cells were observed with regard to all of the plant extracts. Furthermore, M. koenigii methanol extract exhibited promise against human lung cancer cells almost at 80% after 24 h and 90% over 48 h.