About 297,900 Palestinian who lives in 532 residential areas in Area c of Palestine (Jarrar, 2019). Many of them rely on rain harvesting into cisterns during winter to fulfill their needs of drinking water. For instance, in Massafer Yatta, results of water quality from cisterns showed contamination levels that are ranging from 20-100 CFU/100ml. The Bio-sand filter (BSF) has been prompted extensively for water treatment in households in developing countries. In the present study, we investigated the influence of 9 operating parameters on BSF efficiency for Thermo tolerant coliform (TTC). Results analysis showed that five h residence time and media age more than one month were significant with P-values of 0.0439 and 0.0089, respectively. Although the fiveh residence time was insufficient to minimize TTC below the drinking water permissible level of 10 CFU/100ml when influent water had 500 CFU/100ml TTC. In addition, the use of five h residence time may have reduced the filter age and efficiency in removing TTC. Charge volume, turbidity, and using different cistern to fill BSF had no significant influence. The use of sand of 0.18 mm effective size was significant with a P-value of 0.0016. While the temperature itself and inflow rate had no significant influence, the interaction of temperature and size was significant with a P-value of 0.0459. We found that temperature up to 33 °C negatively interfering with BSF’s ability to remove TTC when the BSF has sand size of 0.23mm. The influent TTC count had a significant effect on the effluent BSF TTC with a P-value of 0.0004. The use of 23h residence time was the most influential among all operation parameters with a P-value <0.0001. The study concluded that the most crucial operation parameters for BSF to remove 99.8% TTC are 23h residence time and 0.18mm sand size. The study recommended designing a BSF that produces more than 20L to be used once every 23h and to replace the BSF sand top every four years at maximum.