Cassia fistula L. (Fabaceae) fruit is used traditionally in Nepal as an antipyretic and to treat constipation, while the leaves are used to treat jaundice, piles, rheumatism, ulcers, insect bites, facial paralysis and skin eruptions. C. fistula leaves are important ingredients in Ayurvedic medicine. This study was undertaken to characterize the volatile constituents of C. fistula leaves and to evaluate their antimicrobial and cytotoxic properties. The essential oil from the leaves of Cassia fistula collected from Biratnagar, Nepal, was obtained by hydrodistillation and analyzed by GC-MS. Antimicrobial activities (minimum inhibitory concentration) against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, and Candida albicans, were determined using the microbroth dilution technique, in-vitro cytotoxic activity against MCF-7 human adenocarcinoma cells was determined using the MTT method. C. fistula leaf oil was composed of only seven components, all of which were identified: eugenol (25.0%), (E)-phytol (21.5%), camphor (13.5%), limonene (11.0%), salicyl alcohol (10.4%), linalool (9.9%), and 4- hydroxybenzyl alcohol (8.7%). The leaf oil showed antifungal activity against A. niger (MIC = 78 μg/mL) and C. albicans (MIC = 313 μg/mL), but only marginal cytotoxicity against MCF-7 cells (19.63 ± 11.89% kill at 100 μg/mL). All of the individual essential oil components were screened for activity. Eugenol exhibited antifungal properties (MIC on A. niger = 78 μg/mL) and limonene and phytol were cytotoxic (IC50 = 74.7 and 54.3 μg/mL, respectively).