| Summary: | <i>Cistus ladanifer</i> L. is a plant widely used in folk medicine to treat various illnesses. This study aims to evaluate the effect of the plant flourishing time harvest on the chemical composition and the antimicrobial effect of its essential oil. Chemical analysis of the essential oil was carried out using gas chromatography-mass spectrometry (GC-MS). The antibacterial and antifungal proprieties were tested against four selected bacteria (<i>Staphylococcus aureus</i>, <i>Salmonella Typhi</i>, <i>Escherichia coli</i>, and <i>Acinetobacter baumannii</i>) and nine fungi (Yeasts (<i>Candida tropicalis</i>, <i>Candida glabrata</i>, <i>Candida dubliniensis</i>, <i>Candida</i> sp., <i>Rhodotorula rubra</i>, <i>Cryptococcus neoformans</i>) and molds (<i>Penicillium</i> sp. (P), <i>Fusarium</i> sp. (F), <i>Aspergillus niger</i> (<i>A. niger</i>)), respectively. The essential oil of <i>C. ladanifer</i> demonstrated a powerful antibacterial activity with an inhibition zone of 55 ± 0.22 mm <i>for Staphylococcus aureus,</i> 42 ± 0.11 mm for <i>Escherichia coli,</i> 35 ± 0.27 mm for <i>Acinetobacter baumannii (Full resistant to antibiotics)</i> and 30 ± 0.25 mm for <i>Salmonella Typhi.</i> It also inhibited all tested bacteria at 10 µL/mL. For the antifungal activity test, <i>C. tropicalis</i> and <i>C. neoformans</i> appeared to be the most sensitive strains to the essential oil with an inhibition zone of 13 mm, followed by <i>R. rubra</i> and <i>Penicillium</i> sp. (12 mm), then <i>C. dubliniensis</i> and <i>C. glabrata</i> (11 mm). The chemical analysis of the essential oil by GC-MS revealed that the major components of the essential oil were viridiflorol (17.64%), pinocarveol (11.02%), bornylacetate (9.38%), and ledol (8.85%). <i>C. ladanifer</i> exhibited a remarkable antimicrobial activity that could be more exploited to develop targeted natural remedies against specific diseases.
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