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To mitigate risk to crew health, the microbial surveillance of the quality of potable water sources of the International Space Station (ISS) has been ongoing since before the arrival of the first permanent crew. These water sources have included stored ground-supplied water, water produced by the Shuttle fuel cells during flight, and ISS humidity condensate that is reclaimed and processed. In-flight monitoring was accomplished using a self-contained filtering system designed to allow bacterial growth and enumeration during flight. Upon return to Earth, microbial isolates were identified using 16S ribosomal gene sequencing. While the predominant isolates were common Gram negative bacteria including Ralstonia eutropha, Methylobacterium fujisawaense, and Sphingomonas paucimobilis, opportunistic pathogens such as Stenotrophomonas maltophilia and Pseudomonas aeruginosa were also isolated.