Fishes endemic to freshwaters are strongly influenced by water hardness because of low ionic compositions in the surrounding aquatic media. This compels it to change tissue physiology especially gill and muscle, majorly responsible for calcium metabolism and ionic regulation. Therefore, to understand the interplay between both, the present study evaluated biomarkers in tissues (gills and muscle) of a freshwater fish, Koi carp (Cyprinus carpio var koi) exposed to a sequential increase in water hardness. Glucose, Oxidative stress (Malondialdehyde) and Antioxidant markers (Catalase, Glutathione-S-Transferase and Glutathione) were quantified after 14 days of exposure to a four-fold increase from 75 (Soft - TS), 150 (Moderate - TM), 225 (Hard - TH) to 300 (Very Hard - TV) mg CaCO3/L. Although the fish showed appreciable endurance to all exposures, soft and moderate waters proved harmful to both tissues. Successive increase was observed in the glucose content especially in gills (p < 0.05). Hardness at 75 and 150 mg CaCO3/L was detrimental to both gill and muscle because of elevated MDA concentrations (p < 0.05). Higher CAT, GST activity and GSH levels indicated well coordinated antioxidant enforcement. Overall, the results revealed assorted tissue-specific biomarker responses and more importantly, concentrations < 225 mg CaCO3/L elicited strong oxidative impairment in both gill and muscle.