The use of ‘new psychoactive substances’ appears to be increasingly common. The aim of this study was to examine biological and personality determinants in individuals who choose to use these substances, which may help in the prevention and treatment of psychoactive substance use disorders. The study group consisted of 374 male volunteers; all were users of ‘new psychoactive substances’ (NPS). The NPS users were recruited after they had abstained—for at least 3 months—from any substance of abuse in addiction treatment facilities. The NPS patients and the control subjects were examined by a psychiatrist using the Mini-International Neuropsychiatric Interview (M.I.N.I.), the NEO Five-Factor Personality Inventory (NEO-FFI), and the State-Trait Anxiety Inventory (STAI) scales. The real-time PCR method was used for genotyping. When we compared the controls with the study group, statistically significant interactions were found between DAT1 polymorphism, neuroticism, and NPS use. NPS use and DAT1 polymorphism were associated with a higher level of neuroticism on the NEO-FFI scale. The study group of NPS users showed a higher severity of anxiety symptoms, both in terms of trait and state, compared to the control group. The results may support the idea that neuroticism and anxiety correlate strongly with coping motives for using NPS.