AbstractAlcohol and nicotine are psychoactive substances responsible for serious health consequences. Although the biological mechanisms of alcohol and nicotine have been studied extensively, individual differences in the response to these drugs have received little attention. Here we evaluated gene expression and behavior of bold and shy individuals after acute exposure to alcohol and nicotine. For this, zebrafish were classified as bold and shy individuals based on emergence tests, and then fish were exposed to 0.00, 0.10, and 0.50% alcohol or 0.00, 1.00, and 5.00 mg/L nicotine and their anxiety‐like and locomotor behavior was observed. After behavioral assessment, brain mRNA expression (ache,bdnf, gaba1,gad1b,th1, andtph1) was evaluated. Locomotion patterns differed between profiles depending on alcohol and nicotine concentration. Anxiety increased in shy fish and decreased in bold fish after exposure to both drugs. Alcohol exposure induced an increase intph1mRNA expression in bold fish, whilebdnfmRNA expression was increased in shy fish. Nicotine increasedache,bdnf, andtph1mRNA levels in both profiles, but at higher levels in bold fish. Based on our research, we found that alcohol induces anxiogenic effects in both bold and shy zebrafish. Additionally, shy individuals exposed to a low concentration of nicotine exhibited stronger anxiety‐like responses than their bold counterparts. These findings further support the validity of using zebrafish as a dependable tool for studying the effects of drugs and uncovering the underlying mechanisms associated with individual variations.