Pollutants, such as polycyclic aromatic hydrocarbons (PAHs), e.g., benzo(a)pyrene (BaP), are common components of contaminating mixtures. Such compounds are ubiquitous, extremely toxic, and they pollute soils and aquatic niches. The need for new microorganism-based remediation strategies prompted researchers to identify the most suitable organisms to eliminate pollutants without interfering with the ecosystem. We analyzed the effect caused by BaP on the growth properties of Candida albicans, Debaryomyces hansenii, Rhodotorula mucilaginosa, and Saccharomyces cerevisiae. Their ability to metabolize BaP was also evaluated. The aim was to identify an optimal candidate to be used as the central component of a mycoremediation strategy. The results show that all four yeast species metabolized BaP by more than 70%, whereas their viability was not affected. The best results were observed for D. hansenii. When an incubation was performed in the presence of a cytochrome P450 (CYP) inhibitor, no BaP degradation was observed. Thus, the initial oxidation step is mediated by a CYP enzyme. Additionally, this study identified the D. hansenii DhDIT2 gene as essential to perform the initial degradation of BaP. Hence, we propose that D. hansenii and a S. cerevisiae expressing the DhDIT2 gene are suitable candidates to degrade BaP in contaminated environments.