LGBT individuals experience discrimination in health care settings and report difficulty accessing clinically competent healthcare. This study examined the self-assessed knowledge, clinical preparedness, LGBT health focused education received and attitudinal awareness of health care workers (HCW) (n = 215) toward LGBT patients at an urban hospital in New York City. HCW completed a one-time survey, that included the Lesbian, Gay, Bisexual, and Transgender Development of Clinical Skills Scale. Forty percent of HCW treated LGB patients and 30% treated transgender patients, 11% and 18% reported they were unaware if their patients were LGB or transgender. Seventy-four percent of HCW received less than two hours of formal education in LGBT health. A slight majority of HCW (51%) reported not receiving adequate clinical training to work with transgender clients. Forty-six percent of HCW reported not receiving adequate clinical training to work with LGB clients. A significant difference in LGBT health knowledge, clinical preparedness, and attitudinal awareness was found by LGBT health education received. HCW that reported more LGBT focused health education reported higher basic LGBT health knowledge, felt more clinically prepared, and reported affirming attitudes regarding LGBT patients. This research suggests that more LGBT health focused education of HCW is needed.