The aim of the study was to compare three sampling techniques used in routine diagnostics to identify the microbiota in chronic venous leg ulcers. A total of 46 patients with persisting venous leg ulcers were included in the study. At inclusion, swab, biopsy and filter paper pad samples were collected. After 4 weeks, additional biopsy and filter paper pad samples were collected. Bacteria were isolated and identified at species level by standard methods. The most common bacterial species detected was Staphylococcus aureus found in 89% of the ulcers. No methicillin-resistant S. aureus isolates were found. We did not find any significant differences regarding the bacterial species isolated between the three sampling techniques. However, using multiple techniques led to identification of more species. Our study suggests that it is sufficient to use swab specimens to identify the bacterial species present in chronic wounds, thus avoiding complications during and after biopsy sampling.