Introduction: The attitude of medical students towards homosexuality may affect the quality of care for homosexual patients. This study aimed to describe the attitude of medical students at the National University of Asuncion, Paraguay towards homosexuality. Methods: This observational, cross-sectional study was conducted in June 2016 in consecutive medical students from the National University of Asunción (Santa Rosa del Aguaray branch), Paraguay. The 10-item attitude towards homosexuality scale (EAH-10) was used to assess participants' acceptance/ rejection of homosexuals as individuals, homosexuality as a sexual orientation, and public manifestations of homosexuality. Results: A total of 48 female and 29 male participants (mean age, 21 ± 2 years) were included. Most were Catholic (71.4%), followed by non-Catholic Christian (10.4%), agnostic (9.1%), atheist (2.6%), and other (6.5%). 71.4% reported having at least one homosexual friend. The mean EAH-10 score was 27.23 ± 9.379. 42.9% of participants were indifferent or undecided in their attitude towards homosexuality and 28.6% were discriminatory. Having homosexual friends was associated with a lower EAH-10 score (t =-3.447 [75], p = 0.001). Conclusion: Education about health issues of homosexuals is needed for medical students in Paraguay.