The aim of this work was to investigate and compare the antiproliferative, antioxidant and antibacterial activities of Rosmarinus officinalis essential oil, native to Pakistan. The essential oil content from the leaves of R. officinalis was 0.93 g 100g-1. The GC and GC-MS analysis revealed that the major components determined in R. officinalis essential oil were 1,8-cineol (38.5%), camphor (17.1%), α-pinene (12.3%), limonene (6.23%), camphene (6.00%) and linalool (5.70%). The antiproliferative activity was tested against two cancer (MCF-7 and LNCaP) and one fibroblast cell line (NIH-3T3) using the MTT assay, while, the antioxidant activity was evaluated by the reduction of 2, 2-diphenyl-1-picryl hydrazyl (DPPH) and measuring percent inhibition of peroxidation in linoleic acid system. The disc diffusion and modified resazurin microtitre-plate assays were used to evaluate the inhibition zones (IZ) and minimum inhibitory concentration (MIC) of R. officinalis essential oil, respectively. It is concluded from the results that Rosmarinus officinalis essential oil exhibited antiproliferative, antioxidant and antibacterial activities.