The paper provides an insight into the traditional use of medicinal plant species from the genus Gentiana in the Pirot County in Southeastern Serbia. The ethnopharmacological study was conducted in the form of interviews among the population in four municipalities: Pirot, Babušnica, Bela Palanka, and Dimitrovgrad. Among the population of Pirot County, it was noticed the use of two species from the genus Gentiana: G. cruciata and G. lutea. It was recorded that cross gentian (G. cruciata) has great popularity and versatile aspects on traditional usage, that was mentioned by 53 respondents, and that includes the treatment of cancer diseases, improving the immune system, diabetes, blood purification, for appetite, gastric and duodenal ulcer, for high blood pressure, inflammation, lung diseases, cold, cough, disease prevention, diseases of internal organs, high cholesterol, and leaking breast. The usage of the root of yellow gentian (G. lutea) was mentioned by 30 respondents, and that includes improving the immune system, appetite, for the stomach, blood purification, circulation, as an aphrodisiac, against cancer diseases, diabetes, and gastric and duodenal ulcer. It is important to note that yellow gentian, recorded in the study area, is the first species on the list of illegally collected species in Serbia.