During routine laboratory work, Komagataella phaffii (previously Pichia pastoris) is usually cultivated on rich medium agar plates or in rich or minimal salt-based liquid media, allowing optimal growth within a short time. However, to screen samples containing a diversity of microorganisms (e.g., wastewater or natural environmental samples) for the presence of viable K. phaffii or to aim for the isolation of novel strains, enhanced selective pressure is indispensable. In this chapter, we present a selective medium based on a chemically defined medium composition for K. phaffii in combination with methanol as the sole carbon source and a combination of antibiotics, which together suppress the growth of diverse bacteria. This selective medium is intended to enable an enhanced selective isolation and cultivation of the methylotrophic yeast K. phaffii, especially from samples containing a high microbiological background.