North East region of India suffers from high sulphur coal mine overburden (OB) and plants those survive under such condition have adopted to such harsh environment. Here, we have investigated the effect of coal mine OB substrate on biochemical, physiological and growth of two shrubs Cassia (Cassia sofera L.) and Dhaincha (Sesbania rostrata L.); two tree species Gomari (Gmelina arborea L.) and Sisso (Dalbergia sisso L.); and two monocots Citronella Grass (Cymbopogon winterianus Jowitt) and Lemon Grass (Cymbopogon citratus L). The mine OB was found to be acidic (pH 2.0) with no true soil behavior, 12.5% sulphur and more than double the trace and heavy metals compared to normal control soil. Overall, high lipid peroxidation, membrane damage, peroxidase, glutathione reductase, ascorbate peroxidase, superoxide dismutase enzymes activities along with osmolyte proline and total soluble sugar was found in mine OB plants compared to control plants. While antioxidant activities were higher, nitrate reductase activity was significantly low but reduced leaf area, total shoot and root biomass. However, the responses of antioxidant activities in mine OB plants and control plants did not follow a standard pattern of higher activities in roots followed by old>young leaves or vice versa. Further, in mine OB plants, lipid peroxidation was found significantly higher for young citronella leaves. The levels of higher glutathione reductase was found in roots than the leaves except for old leaves in Gomari while ascorbate peroxidase was found in the leaves than the roots. Overall, all the species showed higher antioxidant enzyme activities, osmolyte accumulation and membrane damage possibly due to acidity and significantly higher limit of trace and heavy metals in mine OB substrate.