The excessive accumulation of inorganic pollutants such as arsenic (As) and fluoride (F) are destructive contaminants for plants. There are some reports about the toxicity mechanisms of individual exposure of As or F. Biochar (BC), carbon-rich organic material, can sequester pollutants and help in increasing growth, nutrient uptake and water holding capacity in soils. However, the defense responses of plants to a combination of two stresses and the interactions between BC and combined exposure of As and F are not explicit. In order to fill this gap, different BC concentrations (BC1; 1 g L−1 and BC2; 5 g L−1) were hydroponically applied to Lemna minor subjected to sodium fluoride (F, 15 mM NaF) and sodium arsenate (As, 100 μM Na2HAsO4). As or F pollutants caused a reduction in the chlorophyll fluorescence (Fv/Fm and Fv/Fo) but BC application under stress conditions improved the fluorescence levels. The photochemical reactions of photosystems and fluorescence kinetics impaired by stress were re-regulated through all BC concentrations. The relative expressions of genes belonging to proteins in PSI-PSII (psaA, psaB, psbA and psbD) were up-regulated in both BCs and As/F treatments. As-treated L. minor had only induction in superoxide dismutase-SOD- and ascorbate peroxidase-APX-, the enhancement in SOD, catalase-CAT-, glutathione reductase-GR- and glutathione S-transferase-GST- was observed under F toxicity. Also, the joint stress (As+F) caused declines in all antioxidant capacities except with SOD activity. Due to the sufficient antioxidant activation, hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) increased in L. minor. While, 1 mg L-1 BC induced the regeneration of ascorbate (AsA) under individual application of As or F, 5 mg L-1 BC increased APX, MDHAR, DHAR, total ascorbate (tAsA), and DHA against F stress. As- and NaF-mediated stress suppressed the nitrogen assimilation of L. minor. Interestingly, the induced accumulation of NH4+ was prevented in BC plus stress-applied plants through glutamate dehydrogenase (GDH) activity rather than glutamine synthetase-glutamate synthase (GS-GOGAT) pathways. All findings indicated that BCs reduced lipid peroxidation and ROS accumulation by modulating electron fluxes and photochemistry in PSI and PSII, gene expression, nitrogen metabolism as well as the antioxidant system under stress conditions (As, F, and As+F).