To assure the quality of the laboratory diagnosis of Buruli ulcer disease; microscopy and PCR were subjected to external quality assurance (EQA). Slides were read by test laboratory staff, followed by blinded re-reading by the controller. Parallel testing of PCR specimens was carried out at the local and external reference laboratory. Slides and PCR specimens with discordant results were subjected to a second reading/testing by the controller to determine the final result. For training purposes, slides and PCR specimens with discrepant results were subsequently re-read/re-tested under supervision at the test laboratory. Microscopy. First reading: concordance rate 82.9%, discordance rate 17.1%, percentage false negatives 27.1% (sensitivity 72.9%), percentage false positives 10.1% (specificity 89.9%). Second reading: concordance rate 97.9%, discordance rate 2.1%, percentage false negatives 4.2% (sensitivity 95.8%), percentage false positives 0.6% (specificity 99.4%). PCR. First testing: concordance rate 87.9%, discordance rate 12.1%, percentage false negatives 8.2% (sensitivity 91.8%), percentage false positives 19.1% (specificity 80.9%). Second testing: concordance rate 96.2%, discordance rate 3.8%, percentage false negatives 4.7% (sensitivity 95.3%), percentage false-positives 2.1% (specificity 97.9%). EQA identified deficiencies in the laboratory performance. Corrective action consisted in on-site training and reduced the number of false-negative and false-positive microscopy and PCR results.