This study aimed to assess the colonization of hotel water systems in central Greece and Corfu by Legionella, and to investigate the association between physicochemical parameters and Legionella colonization. Standardized hygiene inspection was conducted in 51 hotels, and 556 water samples were analyzed for Legionella spp. Free chlorine concentration, pH, hardness, conductivity, and trace metals were defined in cold water samples. The results of inspections and chemical analyses were associated with the microbiological results using univariate and logistic regression analysis. According to the score of the checklist used for the inspections, 17.6% of the hotels were classified as satisfactory, 15.7% as adequate, and 66.7% as unsatisfactory. Moreover, 74.5% of the hotels were colonized by Legionella spp. and 31.4% required remedial measures according to the European guidelines. Legionella spp. were isolated in 28% of the samples. Unsatisfactory results of inspections were associated with Legionella presence (relative risk (RR) = 7.67, p-value = 0.043). In hot-water systems, <50 °C temperatures increased the risk of Legionella colonization (RR = 5.36, p-value < 0.001). In cold-water systems, free chlorine concentration <0.375 mg/L (odds ratio (OR) = 9.76, p-value = 0.001), pH ≥ 7.45 (OR = 4.05, p-value = 0.007), and hardness ≥321 mgCaCO₃/L (OR = 5.63, p-value = 0.003) increased the risk, whereas copper pipes demonstrated a protective role (OR = 0.29, p-value = 0.0024). The majority of the hotels inspected were colonized with Legionella. Supplementary monitoring of the risk factors that were identified should be considered.

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