Introduction. The Candida albicans complex is formed by Candida albicans, Candida dubliniensis, and a biovar of C. albicans named Candida africana. These yeasts are recognized as globally distributed clinical pathogens and share most phenotypic characteristics, which makes their discrimination by conventional methods difficult. Aim. To evaluate the efficacy of different brands of cigarettes in the preparation of tobacco agar, for the differentiation of these related yeasts. Methodology. Tobacco agar was prepared using six brands and four varieties of cigarettes, and 125 clinical isolates previously identified by PCR and Maldi-Tof were used. To determine whether the results of the microbiological tests were associated with similarities in the chemical components of cigarettes, thin-layer chromatography was performed. Results. Candida dubliniensis colonies presented hue differences according to the incubation temperature and the brand or variety of cigarette used, except in the tobacco agar produced with Marlboro Xpress cigarette, where its differentiation was not possible. The chromatograms showed few differences among apolar and medium polarity extract components. Conclusions. Tobacco agar is a low-cost tool used for the differentiation of Candida dubliniensis; however, incubation temperature and cigarette brand affect the performance of the media. No relationship was found between the microbiological results and the chemical similarity of the extracts of the cigarettes by chromatography.