Abstract: Scientific and technological advances in ergonomics/human factors have greatly contributed to the health promotion and comfort of workers in different working environments around the world. Nevertheless, the heat stress is one of the damaging factors to be treated in various industries and jobs. This study was conducted to compare environmental and biological markers of effect of heat stress among the workers engaged in one of the southern seaports of Iran. According to designed methodology, in the hottest hours of the day during the hottest month of the year, the Wet Bulb Globe Temperature (WBGT) was evaluated. Also, biological markers of effect including heart rate and body temperature were studied. To determine worker`s energy demands, the procedure of ISO 8996 was applied. The results showed that during summer, according to environmental monitoring, all of the workers experienced heat stress higher than recommended ACGIH limits. Although, comparison of obtained results from environmental monitoring versus biological monitoring of effect, showed that only 16.25% of exposed population experienced heat stress according to biological markers measurements. According to questionnaire data, acute heat-induced disorders were reported among 23.5% of exposed workers during the study period. Present finding showed that biological monitoring could be regarded as a low cost and more reliable procedure in evaluation and control of heat stress among exposed population for providing comfortable working environment and promoting quality of working life. It may be concluded that in this study, estimation of heat stress using biological monitoring is closer to reality of heat stress in exposed workers, in comparison with WBGT index in which, all of the exposed workers, are experiencing heat stress.