Microbially contaminated metal-working fluid (MWF) can cause respiratory symptoms in exposed workers in the form of exogenous allergic alveolitis/hypersensitivity pneumonitis (HP). The diagnosis of HP is based, among others, on the identification of the culprit and the detection of corresponding specific IgG antibodies (sIgG) in the patient's serum. Commercial antigen tools for the detection of these HP triggers are rarely available; therefore, antigens from contaminated MWF workplace samples were isolated exemplarily for diagnosis of a suspected HP case. Various MWF-specific bacteria were identified in the workplace samples, including Pseudomonas oleovorans, Pseudomonas alcaliphila, Pseudomonas spec., Paenibacillus glucanolyticus, and Corynebacterium amycolatum. The sIgG antigen binding, detected by ImmunoCAP system against MWF antigens from workplace samples and against the identified bacterial antigens, was much stronger in the patient serum compared to selected reference sera. The highest sIgG concentrations in the patient's serum could be determined against Pseudomonas antigens. Inhibition tests showed cross-reactions of MWF and Pseudomonas antigens, whereby the Pseudomonas antigens cross-reacted less with each other. For in-vitro diagnosis in case of suspected HP caused by contaminated MWF, workplace-related antigens are now available.