Abstract Purpose To measure the prevalence of elevated Endotoxin Activity (EA) in a large cohort of patients with Septic Shock (SS), and to assess its value as an early indicator of Gram-Negative (GN) infection, disease severity, and patient risk. Materials and methods Adult patients were enrolled in this observational study if an EA determination was obtained within 24-h from SS onset. Demographic, clinical, and microbiological data were collected. In-hospital follow-up was also conducted. Results A high prevalence of endotoxemia was observed in the 107 subjects included, with 82% of patients showing either intermediate (≥ 0.4 units), or high (≥ 0.6) EA. Patients with positive cultures for GNs showed a higher mean EA (0.63 ± 0.18 vs. 0.53 ± 0.22; p < 0.05). However, the test showed poor accuracy in the identification of GN bacteria as SS causative agents. Significantly higher lactate concentration ( p = 0.006), SOFA ( p = 0.04) and inotropic score ( p = 0.006) were observed in patients with endotoxemia. However, higher EA levels neither influenced mortality, nor length of stay. Conclusions Early after SS onset, patients showed a high prevalence of endotoxemia, particularly those infected with GN bacteria. The EA assay might be a useful marker of disease severity. The complexity of such patients, however, limits EA accuracy in identifying GN sepsis and predicting outcome.