The aim of this study was to determine the total phenolic content, antioxidant and antimicrobial properties of the methanolic extract of Murraya paniculata leaves and its derived fractions as well as characterization the active chemical composition of these extracts by High-Performance Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry (HPLC-ESI-MS). The antioxidant activity was evaluated by using two methods; DPPH free radical scavenging activity and total antioxidant capacity. The identification of certain chemical constituents of M. paniculata methanolic extract, ethyl acetate and butanolic fractions using HPLC-ESI-MS were carried out. The obtained results revealed that the ethyl acetate fraction contains highest contents of phenolic content (607.88 ± 6.87 mg gallic acid eq./g ext.) and flavonoids content (41.53 ± 0.63 mg rutin eq./g ext.). Also, ethyl acetate fraction exhibited the most potent antioxidant and antimicrobial activities.