This study aims to determine prevalence and frequency of use of novel psychoactive substances (NPS) and to identify the factors associated with NPS use in an Italian sample of patients diagnosed with substance use disorder (SUD). Prevalence and correlates of NPS knowledge and use were assessed in 185 patients with SUD in three addiction services (Padova, Belluno, Feltre) in the Veneto region with an ad-hoc designed survey. Two thirds of the samples reported knowing NPS and one third reported using them. NPS were considered by them less dangerous than “regular” substances of abuse (t = 6.06 mean 0.78, p < 0.001). Factors associated with NPS use were youth (OR = 4.81; p < 0.001), studentship (OR = 4.99; p = 0.004), subsequent mental disorders diagnosis (OR = 2.49; p = 0.027), suicide attempt history (OR = 11.67; p < 0.001), home detention (OR = 2.30; p = 0.040), residential care (OR = 5.66; p = 0.002), and polysubstance abuse (t = 8.99 mean 2.65 p < 0.001). NPS use in patients with SUD is highly prevalent, particularly in the youngest age group, and associated with psychiatric comorbidity and worse prognosis. It is crucial to systematically assess NPS use and inform addiction service users with SUD of the toxic and potentially lethal side effects. Mental healthcare professionals working in addiction services should receive education and training. Cohort and longitudinal studies are needed.