The bacterial microbiota plays an important role in the prolonged healing of chronic venous leg ulcers. The present study compared the bacterial diversity within ulcer material from 14 skin graft operations of chronic venous leg ulcers using culture-based methods and molecular biological methods, such as 16S rRNA gene sequencing, fingerprinting, quantitative polymerase chain reaction, and fluorescence in situ hybridization. Each wound contained an average of 5.4 species but the actual species varied between wounds. The diversity determined by culture-based methods and the molecular biological methods was different. All the wounds contained Staphylococcus aureus, whereas Pseudomonas aeruginosa was in six out of 14 wounds. Molecular methods detected anaerobic pathogens in four ulcers that were not detected with anaerobic culture methods. Quantitative polymerase chain reaction was used to compare the abundance of S. aureus and P. aeruginosa at different locations in the ulcers and their numbers varied greatly between samples taken at different locations in the same ulcer. This should be considered when ulcers are investigated in routine clinical care. The differences between the results obtained with culture-based and molecular-based approaches demonstrate that the use of one approach alone is not able to identify all of the bacteria present in the wounds.