Nursing students have limited knowledge of providing quality care to lesbian, gay, bisexual, and transgender individuals. The aim of this study was to explore the psychometric properties of the Nursing Students' Knowledge of and Attitudes toward Lesbian, Gay, Bisexual, and Transgender (LGBT) Health Concerns (NKALH) survey as well as to examine whether sociodemographic variables may affect attitudes toward LGBT individuals. In this cross-sectional study which was carried out from January-March 2022, 258 nursing students from two Greek universities completed the NKALH. For survey translation, the procedure of forward and backward translation was followed. Construct validity, reliability, and internal consistency were explored via confirmatory factor analysis, the test-retest reliability method, and Cronbach's alpha index. The correlation between the dimensions of the attitude subscale was used to explore convergent validity. Analyses were carried out with the Statistical Package for the Social Sciences (SPSS), version 26.0. The level of significance was set at <0.05. Confirmatory factor analysis confirmed that the attitude subscale consists of three dimensions (Comfortable, Responsibility, Willingness to Care). The Pearson test (r) revealed strong correlations between two measurements (Comfortable r = 0.932, p < 0.001, Responsibility r = 0.938, p < 0.001, and Willingness to Care r = 0.915, p < 0.001), indicating good reliability. The Cronbach's alpha index of the total scale was 0.783, highlighting its good internal consistency. Bivariate analysis revealed that sexual orientation, religion, and age are related to knowledge and attitudes toward LGBT individuals. The NKALH survey is a valid and reliable tool to measure the knowledge and attitudes of Greek nursing students on the health concerns of LGBT individuals.