Clusters of holes, such as those in a lotus seedpod, induce trypophobic discomfort. Previous research has demonstrated that high-contrast energy at midrange spatial frequencies in images causes trypophobic discomfort. The present study examined the effects on discomfort of eliminating various spatial frequency components from the images to reveal how each spatial frequency contributes to the discomfort. Experiment 1 showed that eliminating midrange spatial frequencies did not affect trypophobic discomfort, while Experiment 2 revealed that images of holes that consisted of only high-spatial frequencies evoked less discomfort than other images and that images containing only low or midrange spatial frequencies induced as much trypophobic discomfort as did the original images. Finally, Experiment 3 found that participants with a high level of the trypophobic trait experienced stronger discomfort from the original images and the images containing only low or midrange spatial frequencies than participants with a low level of the trypophobic trait. Our findings thus suggest that trypophobic discomfort can be induced by middle and low spatial frequencies.