The immune system plays a critical role in disease prevention via collaboration between the adaptive and innate immune responses. Due to a variety of immune cell subpopulations with different functions including opposing roles, the tumour microenvironment provides opportunities as a therapeutic target for anticancer drug development. Recently the essential oils and their extracts from plants have been gaining attention in cancer research, due to their antioxidant and anti-inflammatory activities making them good candidates as sources of novel anticancer compounds. The essential oil of Melaleuca Alternifolia and its derived products have been previously reported to cause tumour regression, accompanied by an accumulation of neutrophils at treatment sites in various studies. Therefore, the present study was aimed at investigating the immunological responses induced by intratumoural injection of Melaleuca Alternifolia Concentrate (MAC) into subcutaneous tumours of the female transgenic FVB/N c-neu spontaneous murine model of breast cancer, particularly focusing on the role of neutrophils. Following emerging evidence demonstrating the anticancer activity of neutrophils under different stimulants, the present study investigated the immune responses induced by directly injecting MAC into solid tumours of transgenic FVB/N c-neu mice. 4 % v/v MAC was injected directly into the subcutaneous tumours of the female mice and significantly slowed growth of tumours with no observable side effects. The results showed increased levels of neutrophils purified from the treated tumours as compared to vehicle control and untreated control tumours. The results were consistent with previous studies which reported an increased influx of neutrophils into the tumours following MAC injections. The present study further characterised the neutrophils isolated from treated tumours, and the results showed a phenotypic diversity of neutrophils isolated from tumours mostly containing a hypersegmented nucleus constituting the bulk (62.76 %) of the cytotoxic high-density neutrophils. Emerging evidence has indicated the phenotypic diversity and heterogeneity of neutrophils in cancer. Furthermore, this study evaluated the cytotoxicity of the neutrophils isolated from treated tumours, ex vivo. Neutrophils isolated from tumours treated with 4 % v/v MAC showed cytotoxicity towards NeuTL murine breast cancer cells in vitro and effective killing by neutrophils at effector-to-target ratios from10:1 down to 1.25:1 (p<0.001). In summary, the present study found that 4 % v/v MAC impairs the growth of tumours and with no observable toxicity in mice. Also, neutrophils activated in response to treatment of FVB/N tumours with MAC are cytotoxic for NeuTL murine breast cancer cells and are characterised by a hypersegmented nuclear morphology. At present, literature on specific anti-tumorigenic roles played by TANs in the tumour microenvironment is limited. Thus, data from this study contributes towards identification of specific roles and mechanisms underlying the chemoattraction of TANs into the tumour microenvironment and more importantly in response to MAC intervention.