In a previous study, we reported that the sensitivity of PCR targeting the IS2404 insertion sequence of Mycobacterium ulcerans was 98% when it was applied to 4-mm punch biopsy samples of Buruli lesions. Fine-needle aspiration (FNA) is a less traumatic sampling technique for nonulcerated lesions, and we have studied the sensitivity of PCR using FNA samples. Fine-needle aspirates were taken with a 21-gauge needle from 43 patients diagnosed clinically with M. ulcerans disease. Four-millimeter punch biopsies were obtained for microscopy, culture, and PCR targeting the IS2404 insertion sequence. The sensitivity of PCR using samples obtained by FNA was 86% (95% confidence interval [95% CI], 72 to 94%) compared with that for PCR using punch biopsy samples. In this study, the sensitivities of culture and microscopy for punch biopsy samples were 44% (95% CI, 29 to 60%) and 26% (95% CI, 14 to 41%), respectively. This demonstrates that PCR on an FNA sample is a viable minimally invasive technique to diagnose M. ulcerans lesions.