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Infection with bacterial pathogens is examined over the past three decades as a risk factor for the appearance and development of Alzheimer’s disease and Mild Cognitive Impairment (MCI). The aim of this research was the evaluation of two bacterial virulence factors: rhamnolipids (RLs) and flagellin (FliC), as potential biomarkers, in AD and MCI patients. Specifically, RLs and FliC levels were measured with indirect ELISA in blood serum (BS) of 18 AD and 26 MCI patients and in cerebrospinal fluid (CSF) of 54 AD and 47 MCI patients, while 13 and 23 neurologically healthy individuals, respectively, were used as control cohort. RLs were found to be elevated in BS and CSF of AD patients and in MCI patients’ CSF compared to the control group. Remarkable increase was observed in CSF RLs of AD patients in comparison with MCI patients. FliC was increased in AD and MCI patients in CSF samples compared to normal individuals