Background: The source of the pathological changes that occur before an acute Achilles tendon rupture (ATR) is not fully understood. Bacterial DNA has previously been detected in samples from ruptured Achilles tendons, suggesting a pathogenic role of bacteria in ATR. Purpose/Hypothesis: The purpose of this study was to investigate if DNA from bacteria was present in acutely ruptured Achilles tendons. We hypothesized that 20% to 30% of the samples from the rupture site and no samples from healthy tissue would be positive for bacterial DNA. Study Design: Case series; Level of evidence, 4. Methods: This study included 20 consecutive patients scheduled for surgical repair of an acute ATR. Tendon biopsy specimens were taken from the rupture site and from the healthy tendon tissue proximal to the rupture to act as a control. Samples were blinded to the technician and analyzed using polymerase chain reaction targeted to the bacterial 16S rDNA gene and Sanger sequencing to identify the bacterial species present. McNemar test for paired proportions was performed to test for statistically significant differences in the number of samples positive for bacterial DNA between the ruptured and control regions of the Achilles tendon. Results: Of the 20 patients, 1 (5%) had a positive sample with bacterial DNA from the ruptured part of the Achilles tendon. The same patient also had a positive control sample, although with different bacterial DNA. An additional patient had a positive control sample. There was no statistically significant difference in the number of bacterial DNA–positive samples between the ruptured and control regions of the Achilles tendon. The bacteria found (Staphylococcus sp, Micrococcus sp, and Staphylococcus epidermidis) were normal commensal organisms on the human skin. Conclusion: Bacterial DNA was infrequent in tissue from ruptured Achilles tendons and, if identified, likely was a result of contamination. This suggests that bacteria are not involved in the pathological changes occurring before rupture of the Achilles tendon.