Aims: This study aimed to assess the microbial contamination status of drinking water supply among buses at the main bus terminal in Isfahan. Materials and Methods: About 184 drinking water samples were taken randomly from buses arriving at the Sofeh terminal in Isfahan in 2011. For data analysis of one-sample, Kolmogorov-Smirnov test (α = 0.05).and Mann-Whitney U-test with a significant difference (α = 0.05) was used for the effect of setting insulated water tank in cities bus to reduce microbial contamination. Results: Results showed that 15% of all samples were contaminated with total coliforms and 2.5% of samples contaminated with fecal coliforms. The most contaminated samples were related to buses arriving from Shiraz and Booshehr, (fecal MPN~4). About 16.66% of all samples collected from buses without insulated water tanks were contaminated with total coliforms and fecal coliforms. Conclusion: Results indicated that the drinking water available on investigated public transportation was not safe for public health. However, the highest levels of microbial contamination were observed in samples taken from buses without insulated water tanks. In conclusion, a constant and systematic monitoring system is recommended to prevent contamination of drinking water available on buses.