<p>Little is known about the impact of single herbicides and their mixtures on crayfish. Here, we analyzed the variations of metabolism markers in the hemolymph and oxidative balance in the gills and hepatopancreas of crayfish (<i>Parastacus promatensis</i>) exposed to environmentally relevant concentrations of DEZ^® alone (4, 15, and 30 μg/L of i.e., 2,4-D) or its combination with Roundup^® (520 μg/L of i.e., glyphosate). Crayfish were collected from the Garapiá stream in Brazil and then transported in aerated buckets filled with water from the collection site to the culture room. The animals were kept in properly aerated aquariums with controlled conditions of photoperiod, temperature, and pH, and fed raw beef. After 10 days, the crayfish were exposed to 2,4-D alone (D1 = 4, D2 = 15, and D3 = 30 μg/L) or a mixture of 2,4-D with glyphosate (D3 + G). After the exposure period (7 days), we collected hemolymph samples, measured and weighed the animals, and then removed the gills and hepatopancreas. The highest concentration of 2,4-D caused reduced protein levels and increased glucose, lactate, and uric acid levels in the hemolymph. The combination with glyphosate intensified the changes in all metabolites analyzed in the hemolymph. In the gills, all markers of oxidative balance decreased. We observed lipid peroxidation in the hepatopancreas at the highest concentration of 2,4-D despite the increase in the activity of antioxidant enzymes. Therefore, a clear stress response was observed, accompanied by the presence of oxidative damage, which can compromise not only the health but also the survival of these animals.</p> Graphical Abstract <p></p>