Background: We explored the hypothesized importance of early knowledge of microbiological etiology in patients with pleural infection, including comorbidity and treatment factors in the outcome analyses. Methods: Data from the medical records of a large cohort of 437 consecutive patients in 9 hospitals in East-Denmark were included retrospectively. Results: Microbiology, co-morbidity, therapy and outcome are described in detail. Patient groups with microbiology negative and known bacterial etiology had a similar 30-day and 90-day mortality. There were no differences in initial antibiotic treatment regimens, antibiotic treatment duration, rate of intra-pleural fibrinolysis treatment, surgical referral rate, and ICU admittance rate. Patients with microbiology negative etiology were younger (60.8 vs 64.3 years) and fewer had predisposing risk factors (59% vs 71%), but pleural drainage was more often delayed (49% vs 36%). Mortality was similar in patients treated with either of the two nationally recommended initial antibiotic regimens. However, higher 90-day mortality (22.5% vs 9.7%), disease severity (31.5% vs 6.2%), and ICU admittance rate (21.3% vs 2.9%) was observed in a sub-group with initial broad-spectrum treatment compared to patients receiving the nationally recommended initial treatments, irrespective of knowledge of etiology. Several factors correlated independently to 90-day mortality, including age, predisposing risk factors, surgical referral (Odds-Ratios > 1), drainage delay and intra-pleural fibrinolysis (ORs < 1). Conclusions: No difference was found between patients with microbiology negative and known bacterial etiology regarding outcome or treatment parameters. Treatment factors and predisposing factors independently relating to mortality were found in the cohort. Broad-spectrum antibiotics were initially used for treatment of patients with more severe illness and poorer outcome.