Background and Objective: Road transportation and specifically bus travel accounts for the bulk of travel. Contaminated drinking water consumption is one of the issues that threaten health, and always there is pollution possibility in drinking water in buses. This study evaluated the microbial quality of consumed drinking water in buses and also compared it with available standards. Materials and Methods: This study is a cross-sectional study. By considering similar studies, 95% confidence interval and based on the samples size formula for tradition of proportion, 80 buses from 122 buses that entered Bushehr Passenger Depot from other cities or exited it were randomly selected. Over a three-month period, samples were taken from drinking water in these buses. Temperature, pH, residual chlorine, total coliform (TC), and fecal coliform (E. coli) in each sample were measured in accordance with the standard method. Statistical analysis of the data was carried out with SPSS Version 16, and the t-test was performed for statistical difference. Results: The results showed that residual chlorine in 97.5% was zero and pH values of the samples were in the range of 6.8-8.7. TC and E. coli numbers in 12.5% and 8.8% of the samples were higher than those of standards, respectively. Conclusion: Use of ice, washing time of the drinking water tank (daily or weekly), and method of usage and storage of drinking water (with 20-liter tanks) had significant effects on the bacterial loads of the drinking water in the tanks (p value <0.05). Some factors such as tank material, driver's education and age, type and model of the bus, filling time of the tank (daily or weekly), the method of filling and emptying the tank (complete clearing out of the tank or otherwise), and distance had no significant effect on the quality of the drinking water in the buses.