The current study aims to evaluate the antioxidant, antimicrobial and antibiofilm activities of different extracts of Carrot (Daucus carota L.) leaves as well as their total phenolic content (TPC). Also, the chemical profiling of the most promising extract was performed using UPLC-MS/MS analysis in a negative ion mode. In the DPPH assay, the IC50 values were ranged from 28.07 to 71.58 µg/ml, while in the phosphomolybdenum assay the total antioxidant capacity (TAC) values were ranged from 34.0 to 314.67 mg AAE/g dry extract. The antioxidant results were supported by the TPC findings which were ranged from 13.40 to 144.56 GAE/g dry extract. The tested extracts showed variable antimicrobial effects against four pathogenic microbial strains including Staphylococcus aureus (0-15 mm), Escherichia coli (0-13 mm), Candida albicans (0-11 mm) and Aspergillus niger (0-8 mm). While, the antibiofilm inhibitory effect was evaluated against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa with inhibition ratio (%) range of 0-55.26, 0-60.23, 0-40.25, and 0-32.23, respectively. The chemical characterization of the n-butanol as the most potent antioxidant extract led to the identification of 34 compounds and the majority of the identified compounds were categorized as phenolic acids and flavonoids. Following an in silico investigation of the annotated compounds, caffeic and iso-ferulic acids were putatively characterized as probable inhibitors of pyruvate kinase, through which they exert the extract's antibacterial effect. To sum up, Carrot leaves are a promising source of natural antioxidant and antimicrobial compounds that can be used in the development of pharmaceutical industries