Efficient control of pathogenic bacteria, specifically Legionella pneumophila, is one of the main concerns when operating industrial cooling towers. Common practices to limit proliferation involves use of disinfectants, leading to formation of disinfection by-product and increase in water corrosiveness. A disinfectant-free Legionella control method would make the industry more environmentally friendly. A pilot-scale cooling tower (1 m3/h) operated with demineralized water was used to investigate the potential of high-pH conditioning as a disinfectant-free alternative for control of L. pneumophila and other pathogens. One control experiment was performed under standard full-scale operation involving sodium hypochlorite dosage. Thereafter 3 alkaline pHs of the cooling water were tested: 9.0, 9.4 and 9.6. The tests lasted between 25 and 35 days. The cooling water from the basins were analysed for total cell count by flow cytometry, L. pneumophila concentration by plate count and occasional qPCR analyses targeting the mip-gene, bacterial and eukaryotic community analyses with 16S and 18S rRNA gene amplicon sequencing, relative abundance of eukaryotic to prokaryotic DNA by qPCR of the 16S and 18S rRNA gene. The L. pneumophila analyses showed considerable growth at pH 9.0 and pH 9.4 but was maintained below detection limit (< 100 CFU/L) at pH 9.6 without disinfection. Interestingly, the results correlated with the overall abundance of protozoa in the water samples but not directly with the relative abundance of specific reported protozoan hosts of Legionella. The pathogenicity based on 16S rRNA gene amplicon sequencing of the cooling water DNA decreased with increasing pH with a strong decline between pH 9.0 and pH 9.4, from 7.1% to 1.6% of relative abundance of pathogenic genera respectively. A strong shift in microbiome was observed between each tested pH and reproducibility of the experiment at pH 9.6 was confirmed with a duplicate test lasting 80 days. High-pH conditioning ≥ 9.6 is therefore considered as an efficient disinfectant-free cooling tower operation for control of pathogenicity, including L. pneumophila.