Nucleic acid amplification methods facilitate rapid and sensitive detection of clinically relevant fungal pathogens, and can be employed using a variety of patient specimens. However, contamination from various exogenous sources constitutes a serious threat to the validity of amplification-based fungal assays. In this chapter, common origins of fungal contaminants that compromise molecular fungal testing are described, and measures for preventing contamination are proposed. Detailed guidelines for sample handling, reagent selection, contamination screening, and decontamination procedures are provided.