The present study investigates the chemical composition, antioxidant and antimicrobial bioactivities of essential oil and hexane extract from Citrus limon leaves. The isolation of essential oil was carried out using the Clevenger apparatus. The percentage yield of essential oil and hexane extract from Citrus limon leaves was 0.59 and 0.50 %, respectively. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging assay highlighted that Citrus limon leaves essential oil (CLEO) and hexane extract exhibited the significant antioxidant potential of 69.64 and 67.55 %, respectively, compared to the BHT standard. Similarly, a significant inhibition in linoleic acid peroxidation was recorded in both CLEO (81.93 %) and hexane extract (50.34 %). Characterization of chemical constituents in CLEO and extract was executed using GC/MS, where Limonene was detected as a major compound in CLEO (60.52 %) and hexane extract (73.62 %). The haemolytic activity ranged from 2.46 to 5.75 % revealing negligible cytotoxicity of CLEO and hexane extract. In silico studies agree with the in vitro antimicrobial studies, where vinimalol, taraxasterol, and moretenol present in CLEO showed strong interactions/inhibition against dihydroorotase and DNA gyrase from E. coli, and the tyrosyl-tRNA synthetase and DNA gyrase from S. aureus. Based on the current data, it may be concluded that both CLEO and hexane extract possessed significant bioactivities, such as antimicrobial and antioxidant activity, with minimal cytotoxicity.