This paper describes an easy and sensitive flow cytometric assay for assessing the non-specific cytotoxic activity of gilthead seabream (Sparus aurata L.) head-kidney leucocytes against tumor target cells. Concomitantly, the cytotoxic process and the cell types involved were microscopically studied. The assay was based on the consecutive use of two fluorochromes. The targets were preincubated with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and then mixed with effectors. At the end of incubation time propidium iodide (PI) was added. While live effectors were non-fluorescent, live and dead targets retained the DiO (green) fluorescence and non-viable targets and effectors showed PI (red) fluorescence staining. The kinetics of the cytotoxic activity was studied from 10 to 240 min. Lymphocytes, monocyte-macrophages and granulocytes showed non-specific cytotoxic activity, as demonstrated by light and electron microscopy. In conclusion, the technique presented validates the effectiveness of a dual-color flow cytometric assay for assessing the activity of non-specific cytotoxic cells in fish.