As tumour cells use multiple mechanisms to escape from chemotherapeutic drugs, the anti-tumoural activity of naive mouse peripheral blood mononuclear cells was examined in this study, using a mouse melanoma cell subline resistant to doxorubicin (B16R). Multicellular spheroids are known to be the most adapted in-vitro model to mimic solid tumours in vivo and are used to investigate many aspects of tumour biology. For in-vitro studies, murine peripheral blood mononuclear cells recovered by Ficoll gradient centrifugation after caudal puncture were co-cultured with multicellular tumour spheroids of B16R cells. Morphological investigations show that peripheral blood mononuclear cells were gathered and focused around the spheroids after 14 h of co-culture and contacts were established within 32 h. Between 38 and 62 h of co-culture, the size of the spheroids decreased significantly. The peripheral blood mononuclear cells exerted cytolytic effects that correlated with the induction of cell death in spheroids of B16R melanoma cells. Immunological investigations to localize and identify peripheral blood mononuclear cells that exerted anti-tumoural effects have shown that spheroids were deeply infiltrated by monocytes/macrophages at a stage in which a significant cytolytic activity and a strong cell death rate were observed. For in-vivo studies, intratumoural injections of syngeneic naive peripheral blood mononuclear cells were administered. A weak potential in-vivo anti-tumoural effect of these cells was observed (inhibition of B16R melanoma growth by 20–25%) but the median survival time of mice treated with peripheral blood mononuclear cells did not increase compared with untreated control mice. Thus, despite anti-tumoural activities of peripheral blood mononuclear cells against the poorly immunogenic and highly metastatic chemoresistant B16 melanoma cells in vitro, a potential anti-melanoma effect in vivo, if present, did not increase the life span of B16R melanoma-bearing mice.