Pseudomonas aeruginosa is a serious pathogen involved in nosocomial infections. Its pathogenicity is owed to rich production of virulence factors (VIFs) regulated by several complex hierarchical signal systems depending on environmental conditions, medium composition, and the presence of certain active compounds in it. Choline (Ch), which exists in patient tissues, and ethanol (Et), whose consumption aggravates infections, were reported to augment this microbe virulence. The goal of the present study was to show the effect of Et addition to P. aeruginosa cultures in two media (minimal culture medium [MM] and Eagon-Grelet medium [EGM]) in the absence or presence of Ch on its VIF levels. In MM, Et sharply repressed the basal and Ch-induced levels of the P. aeruginosa lectins PA-IL (galactose-specific) and PA-IIL (fucose/mannose-binding) and proteolytic activities, while increasing C-6-HSL (autoinducer), hemolytic phospholipase C (PLC-H), and phosphatase levels. In EGM, it profoundly increased lectin, protease, pyocyanin, rhamnolipid (RhaL), autoinducer, and slightly phosphatase levels, but reduced Ch-induced protease, PLC-H, and acid phosphatase activities, except the short-chain HSL levels, which were increased by Et in combination with Ch. The presented results enlighten part of the complex molecular basis of Et-induced aggravation of P. aeruginosa infections due to increasing the bacterium virulence, which runs in parallel to suppression of the patient's immunity.