Chronic wounds and chronic ulcers are an increasing problem associated with high health care burden and patient burden. The arrested healing of chronic wounds has, in part, been attributed to the presence of biofilms. Substantial research has documented the presence of biofilms in chronic wounds, and many mechanisms of host–pathogen interactions have been uncovered to explain the arrested healing. However, the paradigm of whether biofilms are only observed in chronic infections was recently challenged when biofilms were also observed in acute infections. Here, we characterize the distribution of bacteria in lower leg wounds with particular emphasis on Pseudomonas aeruginosa and Staphylococcus aureus by confocal laser scanning microscopy combined with PNA-FISH staining and routine culture of bacteria. We show that 40% of wounds contained either P. aeruginosa or S. aureus biofilms and demonstrate the presence of scattered single cells in tissues stained with a universal bacterial PNA-FISH probe. Thus, we demonstrate that chronic wounds do not only harbor bacteria organized in biofilms, but also carry populations of scattered single cells and small cell clusters of only a few bacteria. Our findings may influence diagnostic tools being developed to only target biofilms, where single-cell subpopulations thus may be overlooked and possibly lead to false-negative results.