The determination of trace and toxic elements in biological samples (blood, urine and scalp hair samples) of human beings is an important clinical test. The aim of our present study was to determine the concentration of arsenic (As), copper (Cu), cobalt (Co) and manganese (Mn), in biological samples of male production workers (PW) and quality control workers (QW) of steel mill, with aged 25-55 years, to assess the possible influence of environmental exposure. For comparison purpose, the same biological samples of unexposed healthy males of same age group were collected as control subjects. The determination of all elements in biological samples was carried out by electrothermal atomic absorption spectrometry, prior to microwave assisted acid digestion. The accuracy of the As, Cu, Co and Mn measurements was tested by simultaneously analyzing certified reference materials (CRMs) and for comparative purposes conventional wet acid digestion method was used on the same CRMs. No significant differences were observed between the analytical results and the certified values, using both methods (paired t-test at P > 0.05). The results indicate that concentrations of As, Cu, Co and Mn in all three biological samples of the exposed workers (QW and PW) were significantly higher than those of the controls. The possible correlation of these elements with the etiology of different physiological disorders is discussed. The results were also demonstrated the need of attention for improvements in workplace, ventilation and industrial hygiene practices.