Urticaria is a skin disorder characterized by erythematous, edematous, and pruritic lesions. Intestinal microorganisms can trigger various immunological responses, and Blastocystis has been suggested to affect gut-associated lymphoid tissue homeostasis and induce allergic reactions. This study aimed to evaluate the effect of Blastocystis on the intestinal microbiota in patients with urticaria. A total of 33 patients diagnosed with urticaria and 34 healthy controls were included. Independent sample t-tests, Welch’s t-tests, or Mann–Whitney U tests were applied to assess differences in the Shannon, Simpson, and Chao-1 indices between groups. Significant differences were observed in Proteobacteria (p = 0.015), Bacteroidetes (p = 0.008), Escherichia (p = 0.005), Phocaelcola (p = 0.043), and Prevotella (p = 0.047) between the urticaria and control groups. Bacteroidetes (p = 0.003) and Phocaelcola (p = 0.032) also differed significantly between samples with and without Blastocystis. Overall microbiota composition showed a significant difference between Blastocystis-positive and -negative samples (p = 0.009). The Firmicutes/Bacteroidetes ratio was 4.1 in healthy controls and 6.4 in urticaria patients. In conclusion, both urticaria and Blastocystis infection significantly influence intestinal microbiota composition, suggesting a potential interaction between Blastocystis colonization and host immune regulation in urticaria.