Pygmy waterlily (Nymphaea tetragona, Nymphaeaceae) is a perennial aquatic herb with floating leaves and beautiful flowers. Leaf spot on pygmy waterlilies appeared in 2009 at several locations in Korea, e.g., Suwon in 2009, Yangpyeong in 2011, and Seoul in 2014. The leaf spots were circular to irregular, small, and brown in the center with a dark brown margin. The spots later became milky white. The causative agent of the leaf spot was identified as Pseudocercospora nymphaeacea. Morphological observations and multigene phylogenetic analyses of the internal transcribed spacer (ITS), the partial translation elongation factor 1-alpha (TEF-1α), and actin (ACT) regions are provided. The pathogenicity test was conducted twice with similar results, which fulfilled Koch’s postulates. To our knowledge, this is the first report on P. nymphaeacea infection of N. tetragona.