Application of biomarkers is an effective approach for a better understanding of varying toxicity in aquatic organisms during the seasonal and diurnal changes in the natural environment. This report describes the toxicity of sub-lethal concentrations of triclosan (TCS) at different pH (6.5, 7.5 and 8.5) based on selected biomarkers related to oxidative stress, metabolism and genotoxicity in Pangasianodon hypophthalmus. The 96 h LC of TCS for P. hypophthalmus was lower at pH 6.5 when compared to higher pH. The sub-lethal concentration of TCS exhibited a significant decrease in hematological parameters related to complete blood counts except for total leukocyte count (TLC), mean cell haemoglobin concentration (MCHC) and red cell distribution width (RDW). Multivariate data analysis showed a significant interaction of TCS and pH in metabolizing enzymes like glutamic oxaloacetate transaminase (GOT), glutamic pyruvic transaminase (GPT), Lactate dehydrogenase (LDH), antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione-s-transferase (GST) and neurotransmitter enzyme acetylcholinesterase (AChE). A significant increase in DNA damage and micronuclei frequency in liver and blood cells of TCS exposed fish at pH 6.5 indicate that the TCS exposure has pronounced effects on genetic materials. The findings of present study establish that enzymes like SOD, LDH, GOT, AChE, DNA damage and micronuclei frequency can be successfully deployed as biomarkers for the assessment of toxicity of TCS in fish.