Background: Fentanyl has become increasingly present in the illicit drug supply, including in cocaine. Interventions that target non-opioid use are a necessary component of overdose prevention due to the increased presence of fentanyl. Objectives: To assess management and staff reception to overdose prevention messaging and access to naloxone in New York City nightlife venues. Methods: Between June and August 2019, we conducted a public awareness intervention in 75 nightlife venues (bars, nightclubs and music venues) to visibly display coasters and posters containing overdose prevention messaging for 6 weeks. As part of the intervention, staff were offered overdose education and a naloxone kit. A post-intervention survey assessed reception of intervention materials, staff and patron responses to intervention messaging and staff awareness of fentanyl, naloxone and access to naloxone. Results: Forty-one venues displayed posters and coasters and staff at 28 of these venues (68%) completed naloxone training. Respondents reported learning about naloxone and fentanyl through the intervention, and a majority of staff (92%) were receptive to the intervention's messages. Partnerships with community-facing stakeholders and earned media facilitated the reach of the intervention. Conclusion: Venue management and staff were receptive to the intervention, its messaging and keeping a naloxone kit on the premises. Overdose prevention efforts in nightlife venues should consider low-cost strategies such as disseminating intervention materials through television, print and social media, collaborating with community partners to enhance venue recruitment and providing venues with naloxone training and communal kits. Further research is needed to explore retention of overdose prevention messaging and subsequent short- and long-term behaviour change.