In the present study, three Libyan medicinal plants Juniperus phoenicea, Pistacia lentiscus and Cupressus sempervirens were investigated for their chemical constituents. Studies were conducted to determine the active chemical constituents of alcoholic extracts by way of phytochemistry and to evaluate its in vitro antimicrobial activity, using the disc diffusion technique as described by the National Committee of Clinical Laboratory Standard (1993). The alcoholic extracts of all plants produced a dose dependent zone of inhibition in all the organisms tested (Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae). Due to their medicinal value and relative abundance, it was decided to investigate the flavonoids from these plants. The present investigation has led to the isolation of five flavonoids from an ethanol extract prepared from the leaves, barks and roots of these plants by preparative thin-layer chromatography technique. Their structures were determined through a combination of spectrometric techniques including IR, UV, 1D and 2D NMR and high-resolution electrospray mass spectrometry.