Studies have shown that exposure to either fungicide difenoconazole (DIF) or salt (NaCl) alone can cause phytotoxicity in plants, but it remains uncertain whether co-exposure to these two xenobiotics, which often co-occur in the agroecosystem, can also induce phytotoxicity. This research aimed to study the phytotoxicity and underlying mechanisms of co-exposure to DIF (field recommended doses (0.5 L ha− 1) and NaCl (150 mM) in tomato seedlings. The results showed that exposure to DIF and/or NaCl causes phytotoxic effects in tomato seedlings, including a decrease in fresh weight and length of shoots and roots, damage to chlorophyll pigment, and induced oxidative stress in the leaves. Interestingly, combined exposure to DIF and NaCl exhibited synergistic effects on shoot and root biomass inhibition. Antioxidant defense analyses revealed that the DIF and/or NaCl exposure altered the activities of enzymes involved in the H2O2 scavenging (ascorbate peroxidase and catalase), and in xenobiotic detoxification (glutathione-s-transferase and peroxidase) in leaf tissues. Interestingly, combined exposure to DIF and NaCl markedly enhanced detoxifying enzymes, thereby enhancing xenobiotic biotransformation. In addition, DIF and/or NaCl exposure enhanced proline accumulation, and altered the thiols profile (reduced glutathione) content, while stimulating the phenylpropanoid pathway (phenylalanine ammonia-lyase activity) to produce secondary metabolites such as polyphenols and flavonoids. As pioneer research to highlight the phytotoxicity induced by co-exposure to DIF and NaCl in tomato seedlings, these results provide new insights into physio-biochemical responses of non-target plants to DIF and NaCl co-contamination and shedding light on the ecological risks of pesticides and salt exposure in agroecosystems.