The present study was designed to see the effect of exogenous nitric oxide (NO) and abscisic acid (ABA) and their interaction on physiological and biochemical activities in leaves and roots of two Indian mustard (Brassica juncea) cultivars [cv. Pusa Jagannath (PJN) and Varuna (VAR)] exposed to polyethylene glycol (PEG)-induced drought stress. Seven days old hydroponically grown seedlings were treated with PEG (10%), sodium nitroprusside, a NO donor [NO (100 μM)] and abscisic acid [ABA (10 μM)], using different combinations as: Control, ABA, NO, PEG, PEG + ABA, PEG + NO, and PEG + NO + ABA. Results revealed that in response to PEG-induced drought stress leaf relative water content, chlorophyll, carotenoid and protein content decreased with increased production of O, MDA, HO, cysteine content and non-enzymatic antioxidants (including proline, flavonoid, phenolic, anthocyanin, and ascorbic acid), whereas, the enzymatic antioxidants (including SOD, CAT, APX, GR) showed the response range from no effect to increase or decrease in certain enzymes in both Brassica cultivars. The application of NO or/and ABA in PEG-stressed cultivars showed that both enzymatic and non-enzymatic antioxidants responded differently to attenuate oxidative stress in leaves and roots of both cultivars. Overall, PJN had the antioxidant protection mainly through the accumulation of non-enzymatic antioxidants, whereas VAR showed tolerance by the enhancement of both enzymatic and non-enzymatic antioxidant activities. Altogether, the study concluded that the independent NO and its interaction with ABA (PEG + NO and PEG + NO + ABA) were much effective than independent ABA (PEG + ABA) in lowering PEG-drought stress in Brassica cultivars.