We aimed to investigate the polluted working environment triggers oxidative stress and alter enzymatic antioxidant activity by a short-term interval. The experimental study, performed in 2014, involved 94 workers from the Velenje Coalmine in Slovenia, arranged into three groups according to a number of consecutive working days in a mineshaft, supported by a control group. Levels of the antioxidant enzymes (GPx, CAT, SOD) together with TAC (the combined effect of all antioxidants) and 8-isoprostane (a biological marker of oxidative stress/damage) were measured in human plasma. Workers occupationally exposed for three consecutive working days had significantly increased 8-isoprostane biomarker, a parameter of oxidative stress ( <0.001). The antioxidant levels of TAC ( <0.001), CAT ( <0.001) and SOD ( <0.001) were all significantly decreased compared to a control group. Workers in polluted working environment had significantly increased oxidative stress and altered antioxidant activity already on a third consecutive working day.