Introduction: There is growing interest in the public health and transport sectors in research into exposure to biological hazards, considering not only the risks arising from inter-human contagion, but also those related to exposure to the flight environment itself. The aim of this paper is to report data from an investigation into the water and air-conditioning systems of commercial aircraft for the presence of Legionella contamination, with a total of 645 water samples taken during the period 2007–2021. Methods: The investigation involved 126 aircraft of six different commercial aircraft types: MD80, Airbus A320 F, Embraer 175/190, AIRBUS A330, Boeing 767 and Boeing 777. Water samples were taken from the water systems (toilet taps, galley and boilers). Each sample was preliminarily subjected to an evaluation of the following parameters: temperature, pH and residual chlorine. The ScanVit® Legionella kit was used for bacteria detection and enumeration. Results: Samples were considered positive if the number of colony-forming units/liter (CFU/L) was >100. For the entire observation period, 45% of the investigated aircraft tested positive. Regarding the overall number of samples analyzed, 68.4% (441/645) were below 100 CFU/L, and thus within the limits allowed by the Italian Guidelines. Conclusions: Water system contamination with Legionella in the air transport field is a real public health issue that should not be underestimated given the heavy passenger traffic. Infection should be considered an occupational risk to which crew members are exposed.