Amount of drinking water services through drinking water depots in Kupang City is increasing in avarage of 1.44 every year since 2010, meanwhile there is no guarantee that refill drinking water quality meets any requirement every time. Results of refill drinking water sample in Kupang City in 2013 showed the water was 37.5% contaminated by microbes. This study aimed to analyze microbial contamination and determine determinants of Escherichia coli (E. coli) and total Coliform on refill drinking water. This study used cross sectional design on January - March 2015. The population was 51 depots determined using total sampling technique. Data analysis was conducted in univariate, bivariate using simple logistic regression test and multivariate using multiple logistic regression test. Results showed drinking water contaminated by microbes worth 26 depots (51%), by E. coli 33.33% and by total Coliform 51%. Microbial contamination determinants using bivariate were knowledge (p value = 0.01) and behavior of operator (p value = 0.05). Meanwhile, microbial contamination determinants conducting multivariate were knowledge (p value = 0.026), hygiene of operator (p value = 0.05) and depot sanitation (p value = 0.044). Most dominating variables influencing microbial contamination are knowledge, operator's hygiene and depot sanitation.