Background and objective Substance use disorders pose significant global public health challenges, with India being no exception. Bihar, one of India's most populous states, implemented alcohol prohibition in April 2016 to address the adverse effects of alcohol abuse. However, the impact of this policy on overall substance use behavior among patients in healthcare settings remains to be explored. This cross-sectional study aimed to evaluate the changing trends in substance use behavior among patients in the tertiary care setting following the prohibition of alcohol use in Bihar. Methods A total of 372 patients diagnosed with substance use disorders were recruited from tertiary care facilities in Bihar. Data on demographic characteristics, types of substances used, frequency and quantity of use, reasons for use, and awareness of prohibition laws were collected through structured interviews and reviews of medical records. Descriptive and inferential statistics were used for data analysis. Results The majority of the participants were male (n = 346, 93.01%), with a mean age of 38.5 years. While tobacco use remains stable, there are significant increases in opioid and cannabis consumption post-prohibition, highlighting unintended consequences (p-values - opioids: 0.008, cannabis: 0.021). Additionally, heightened daily and weekly substance use after prohibition is evident (p-values: daily: 0.008, weekly: 0.021), emphasizing the necessity for nuanced policy considerations. Reasons for substance use, including coping with stress and peer pressure, showed significant differences before and after the prohibition (p<0.05). Moreover, awareness of alcohol prohibition laws increased significantly after the implementation of the prohibition (p = 0.003). Conclusions Our findings suggest that while alcohol prohibition in Bihar did not significantly lead to any changes in terms of the types of substances used among patients in tertiary care settings, it did influence the frequency and quantity of tobacco and cannabis consumption. Increased awareness of prohibition laws underscores the importance of policy enforcement and public education initiatives in addressing substance use behavior., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Institutional Research Committee, All India Institute of Medical Sciences, Patna issued approval AIIMS/Pat/IRC/2020/349. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Prasad et al.)