Fluid from a post‐operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse‐phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti‐Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1–11,1 – 15 and 1 – 16, intact thymosin β‐4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL‐39 and its precursor(s). The HPLC‐separated blister fluid was extremely rich in anti‐Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50‐fold differences in anti‐Bm activities and more than 10‐fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.