Background: Lesbian, gay, bisexual, and transgender (LGBT) populations are more likely to misuse alcohol and other drugs (AOD), compared to the general population. However, LGBT engagement with AOD treatment is often precluded by insensitivity and misunderstanding of LGBT issues. These treatment barriers may be a consequence of either worker attitudes, organizational factors or a combination of both. Few studies have compared service context as an impediment to AOD treatment. Objectives: This pilot study sought to examine and compare staff attitudes, knowledge and awareness of LGBT issues in two state-wide AOD services within Australia. One organization was a government service, whilst the other was faith based. Methods: A cross-sectional study of a convenience sample (N = 130) of workers employed in a state-wide government AOD service (n = 65), and a state-wide non-government service (n = 65) was conducted. Participants self-completed a questionnaire comprising tools previously used to assess staff attitudes, knowledge and awareness of LGBT issues. Results: Few significant differences in attitudes and awareness of LGBT issues between government and non-government respondents were found. Nearly all respondents were supportive of LGBT persons irrespective of organizational context, with a small number of negative views. Although most respondents demonstrated awareness of organizational policies and practices relating to LGBT clients, many were “unsure” or “neutral” of what these might be. Conclusion: It is confirming that the majority of staff report supportive attitudes towards LGBT clients. Findings suggest that organizations need to continue to take leadership to strengthen organizational training and capacity to deliver LGBT friendly AOD treatment practices.