The aim of this study was to identify the pathogens potentially involved in parietal fibrinous peritonitis (PFP). PFP is a complication of laparotomy in cattle, characterized by an accumulation of exudate inside a fibrinous capsule. We have studied 72 cases of PFP in Belgian blue cows, confirmed by a standard diagnostic protocol. Blood was collected to evaluate the presence of antibodies for Mycoplasma bovis (M. bovis) , Coxiella burnetii (C. burnetii) and Bovine Herpesvirus 4 (BoHV4 ) by enzyme-linked immunosorbent assays. Peritoneal exudate was obtained from the PFP cavity to perform bacteriological culture, and to identify the DNA of M . bovis , C . burnetii and BoHV4 using real time polymerase chain reaction (qPCR). Bacteriological culture was positive in most peritoneal samples (59/72); Trueperella pyogenes ( T. pyogenes ) (51/72) and Escherichia coli ( E. coli ) (20/72) were the most frequently identified. For BoHV4 , the majority of cows showed positive serology and qPCR (56/72 and 49/72, respectively). Contrariwise, M. bovis (17/72 and 6/72, respectively) and C. burnetii (15/72 and 6/72, respectively) were less frequently detected ( p < 0.0001). Our study proves that PFP can no longer be qualified as a sterile inflammation. Moreover, we herein describe the first identification of BoHV4 and C. burnetii in cows affected by PFP.