Microbiological tests for diagnosis of acute meningococcal disease are important for the clinical management of patients with this often-fatal illness, but cultures are frequently negative after antibiotics have been administered. Retrospective studies suggest that examination of skin biopsies may aid a rapid diagnosis and that cultures of skin biopsies are often positive even after antimicrobial treatment has commenced. This prospective controlled study aimed to assess the diagnostic value of skin biopsy compared with investigations of blood and cerebrospinal fluid (CSF) in patients with skin lesions and presumed meningococcal disease. A total of 43 patients, 31 with suspected acute meningococcal infection and 12 controls, were included. All skin biopsies were investigated by Gram stain and routine microbiological culture. In 25 patients, meningococcal infection was diagnosed microbiologically. The clinical diagnosis was meningococcal meningitis in 8 patients, meningococcal sepsis in 11 patients, and a combination of both in 6 patients. The sensitivity of cultures of blood, CSF, and skin biopsies was 56%, 50%, and 36%, respectively. When culture and Gram stain were combined, positive results were obtained in 56%, 64%, and 56%, respectively. There was no correlation between the diagnostic yield of skin biopsies and previous antibiotic treatment. In 14 patients, the diagnosis was based exclusively on one positive sample: CSF in 7 (28%) patients, blood in 4 (16%) patients, and skin biopsy in 3 (12%) patients. The sensitivity of skin biopsies was highest in patients with the least extensive skin lesions. Specificity was 100%. Microbiological investigation of skin biopsies increased the diagnostic yield and could be considered a component of the routine diagnostic work-up in patients with suspected meningococcal infection, even after the initiation of antimicrobial treatment.