Background Frequencies of polymicrobial infection and pathogens evidenced in course of infected nonunion treatment are largely unknown. Therefore, this study aims at investigating microbial patterns in infected nonunions. Methods Surgically treated patients with long bone infected nonunion admitted between January 2010 and March 2018 were included in the study. Microbiological culture and polymerase-chain-reaction results of tissue samples of initial and follow-up revision surgeries were assessed and compared with patient and treatment characteristics. Results Forty two patients with a mean age of 53.9 +/- 17.7 years were included. In six patients (14.3%) polymicrobial infection was evident. A change of pathogens evidenced in course of the treatment occurred in 21 patients (50%). In 16 patients (38.1%) previously detected bacteria could be determined by microbial testing after further revision surgery.Staphylococcus aureuswas most often detected (n = 34, 30.6%), followed byEnterococcus spp. (n = 25, 22.5%) andStaphylococcus epidermidis(n = 18, 16.2%). FiveStaphylococcus aureuswere resistant to methicillin (MRSA). In patients without polymicrobial infection or further germ detection in course of the treatment, 86.4% of the infections were due toStaphylococcusspp.. Infections due toStreptococcusspp. and gram-negative bacteria were only present in patients with polymicrobial infection and germ-change in course of the treatment. Conclusion A low rate of polymicrobial infections was evidenced in the present study. Germ-change often occurs in course of revision surgeries. Prospective studies with more sensitive diagnostic tools are necessary to elucidate the therapeutical relevance of microbiological testing results for surgical as well as medical treatment in infected nonunions.