Pulicaria odora is a plant widely used in traditional medicine in Algeria. This work focuses on the study of total polyphenol content, flavonoid content, antioxidant activity, and antimicrobial activity. Polyphenols in P. odora leaves have been identified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC/ESI-MS/MS). The results showed a yield of 17.1%. Total content of phenolic compounds expressed at 2.77 mg Eq AG /g ES, and flavonoids evaluated at 0.70 mg Eq Q/g ES. The LC/ESI-MS/MS of the hydromethanolic extract revealed the existence of twelve phenolic compounds. The antioxidant activity evaluated using the DPPH free radical reduction method and FRAP, revealed that extract of P. odora marked a powerful antioxidant potential compared to the standard ascorbic acid. The antimicrobial activity of the extract was tested on six strains of bacteria and three fungal strains by a biological test of an antibiogram, followed by the determination of the minimum inhibitory concentration (CMI). In addition, P.odora extract showed a good inhibitory effect on bacterial and fungal growth against most tested strains, with inhibition areas ranging from 23 mm to 06 mm on bacteria and the fungal strain. These results support the notion that P. odora extract can be used as natural antimicrobial role in pharmaceutical and food preservation systems.