This study aimed to assess the effect of smoking on the biodiversity of the oral fungal microbiota of healthy young subjects, using an improved culture method that assesses both total and pathogenic viable fungi. Forty individuals (20 smokers and 20 non-smokers) were selected. All individuals presented fungal growth (100% for molds and 92.5% for yeasts), a prevalence higher than previously reported. The most commonly occurring molds were Penicillium sp., Aspergillus sp., and Cladosporium sp. Smokers presented significantly higher levels of yeasts and pathogenic molds than did non-smokers. No differences in fungal prevalence and diversity were observed in smokers and non-smokers following a 30-wk observation period. In conclusion, tobacco smoking may alter the oral mycobiota and facilitate colonization of the oral cavity with yeasts and pathogenic molds. The effect of chronic fungal colonization on the oral health of tobacco smokers cannot be neglected.