•Combined stress greatly enhanced lipid peroxidation in 84-S in contrast to M-503.•Heat stress did not have any additional effect on that of only drought in both cultivars.•Tolerant M-503 showed a higher increase in proline content under combined stress.•The sensitivity of 84-S may be related with a decreased activity of CAT and POX. Crop losses due to combined drought and heat is predicted be greater in the future especially due to climate change. Understanding underlying mechanisms under drought and heat combination will be crucial for the selection and breeding of tolerant varieties. The objective of this study was to assess the physiological and biochemical responses of two cotton cultivars (84-S and M-503) differing in drought tolerance to the combined effects of drought and heat. The relative growth rate (RGR) of the cultivars was decreased by 62.9% in drought sensitive 84-S and reduced by 34.58% in drought tolerant M-503 due to the combined drought and heat stresses. Combined stress also enhanced lipid peroxidation (TBARS) by 170.24% and 21.9% in 84-S and M-503, respectively which suggest that drought sensitive 84-S is more sensitive to combined stress than M-503. This sensitivity to combined stress of 84-S was associated with decreased activities of catalase (CAT) and peroxidase (POX) as compared to its control, resulting in higher H2O2 accumulation and oxidative stress induced lipid peroxidation. On the other hand, a higher combined stress tolerance of M-503 was associated with its ability to maintain constitutive activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) and induced CAT and POX. The proline content of drought resistant M-503 was greatly enhanced under drought and the combination of drought and heat treatments as compared to 84-S. To the best of our knowledge, this is the first study conducted on the activities of antioxidant enzymes of cotton under drought and heat combination.