The study evaluated the effects of two selected chemicals; methylene blue and saline on bacterial load in transport-water containing monosex tilapia (Oreochromis niloticus) fry. Fish fry was collected from a private hatchery located in Trishal upazilla, Mymensingh. A total of approximately 550 fry were added in each polyethylene bag containing water and transported to the Fish Disease Laboratory of the Department of Aquaculture, Bangladesh Agricultural University (BAU) for analysis. Two different concentrations of chemicals like 1 and 2 ppm methylene blue, 1 and 2 ppt saline and combination of the both were used. Samplings were done at 0, 4, 8 hours interval to determine the bacterial load in transport-water. Both methylene blue and saline were found effective in reducing bacterial load in water during transportation. The combined use of saline and methylene blue showed better result than the chemicals used individually in terms of final bacterial load. A gradual increase in bacterial load with time was found in control water having no chemicals (T₀). In T₁ (1 ppm methylene blue used) and T₂ (2 ppm methylene blue used) the mean bacterial loads were 1.03±0.5×10⁷ cfu/ml and 5.00±2×10⁷ cfu/ml respectively. Similarly mean bacterial loads of 4.48±1.5×10⁶ cfu/ml and 3.43±2.7×10⁶ cfu/ml were obtained with 1 and 2 ppt saline (T₃ and T₄) respectively. Whereas combined use of 1 ppm methylene blue and 1 ppt saline (T₅) end up with the mean bacterial load of 2.94±2.5×10⁶ cfu/ml after 8 hours of exposure. It was 2.95±2.7×10⁶ cfu/ml when the dose was doubled (T₆). The highest percentage of mortality was found in control water in every sampling period. Water quality parameters like temperature, pH, and dissolved oxygen decreased after 8 hours of transportation. The experiment revealed that the combined low dose of methylene blue and saline can be recommended for transportation of tilapia fry.