Background: A variety of methods exist to take samples from surgical site infections for cultivation, however, an unambiguous and suitable method has not yet been defined. The aim of our retrospective non-randomized study was to compare two non-invasive techniques of sampling material for microbiologic analysis in surgical practice. We compared bacteria cultured from samples obtained with the use of the swab technique, defined in our study as the gold standard, with the indirect imprint technique. Methods: A cotton-tipped swab (Copan, Brescia, Italy) was used; the imprints were taken using Whatman no. 4 filter paper (Macherey-Nagal, Duren, Germany) cut into 5 · 5 cm pieces placed on blood agar in a Petri dish. To culture the microorganisms in the microbiologic laboratory, we used blood agar, UriSelect 4 medium (Bio-Rad, Marnes-la-Coquette, France), and a medium with sodium chloride (blood agar with salt). After careful debridement, a sample was taken from the incision surface by swab and subsequently the same area of the surface was imprinted onto a filter paper. The samples were analyzed in the microbiologic laboratory under standard safety precautions. The cultivation results of the two techniques were statistically processed using contingency tables and the McNemar test. Those samples that were simultaneously cultivation positive by imprint and negative by swabbing were processed in greater detail. Results: Over the period between October 2008 and March 2013, 177 samples from 70 patients were analyzed. Sampling was carried out in 42 males and 28 females. One hundred forty-six samples were from incisions after operations (21 samples from 6 patients after operation on the thoracic cavity, 73 samples from 35 patients after operation on the abdominal cavity combined with the gastrointestinal tract, 52 samples from 19 patients with other post-operative site infections not included above) and 31 samples were from 11 patients with no postoperative site infection. One patient had a sample taken both from a post-operative and a non-postoperative site infection. Coincidently, the most frequent cultivation finding with both techniques was a sterile one (imprint, 62; swab, 50). The most frequently cultivated microorganism after swabbing was Pseudomonas aeruginosa (22 cases), compared with Escherichia coli when the filter paper (imprint) was used (31 cases). The imprint technique was evaluated as more sensitive compared to swabbing at the level of statistical significance p = 0.0001, and k indice to evaluate the concordance between the two techniques was 0.302. Of the 177 samples there were 53 samples simultaneously sterile using the swab and positive in the imprint. In 3 samples colony forming units (CFU) were not counted; 22 samples were within the limit of 0–25 · 101 CFU/cm2, 20 samples within the limit of 25 · 101–25 · 102 CFU/cm2, 5 within the limit of 25 · 102–25 · 103 CFU/cm2, and 3 of more than 25 · 104 CFU/cm2. Conclusions: The hypothesis of swabbing as a more precise technique was not confirmed. In our study the imprint technique appeared to be more sensitive than swabbing and the strength of agreement was considered as fair. We obtained information not only on the type of the microorganism cultured, but also on the number of viable colonies per square unit, expressed in CFU/cm2.