The development of non-toxic, novel anti-tumor alternatives that target key hallmark events of tumor progression is of a high priority for cancer therapy. Natural compounds, such as Essential oils (EOs) derived from plant extracts are a mixture of chemical components known for their diverse pharmacological properties, including anticancer potential. For this purpose, we investigated the antitumor activity of Eucalyptus globulus essential oil (EEO) and its major constituents against colorectal cancer cells in vitro. EEO significantly reduced the viability of colon cancer LS174 cells, induced caspase-dependent apoptosis and triggered cell cycle arrest by modulating the expression of several effectors involved in these processes. Mechanistically, EEO exhibited its activity by targeting p38, SAPK/JNK, ERK1/2, and AKT kinases in LS174 cells. Considering the pivotal role of p53 status in mediating the response to anticancer therapies, we further investigated the effects of Eucalyptol, 3-Cyclohexene-1-methanol, α-Pinene, and α-Terpineol, identified as major components of EEO, on the viability of human colon adenocarcinoma LS174 (wild type p53) and HT29 (mutant p53) cell lines. Interestingly, we highlighted for the first time that 3-Cyclohexene-1-methanol exhibited the most anti-proliferative activity against both tumor cells irrespective to their p53 status. It exerted its effect by inducing apoptotic cell death, disturbing cell cycle progression along with reducing the phosphorylation of key components of the proliferation and survival pathways p38, ERK1/2, and AKT kinases. Our results suggest that Eucalyptus essential oil and its component, 3-Cyclohexene-1-methanol represent promising multi-targeting candidates for colorectal cancer therapy.