Fish transportation is a regular farming and experimental practice that mainly results in stress conditions that may extend to induce a high mortality rate. In the present experimental simulation, Oreochromis niloticus were transported for 2 h and maintained for another 6 h in transportation containers to assess the effect of tranquilization with tricaine methanesulfonate (MS-222) and/or the addition of iodine on mortality rate and restoring the normal physiological functions. Experimental fish were divided into four groups: group 1 (control) was transported in farm water without MS-222 or iodine, group (2) was transported in farm water supplemented with 40 mg/L of MS-222, fish in group (3) were transported in farm water supplemented with 40 mg/L of MS-222 + 10 ppm iodine, and fish in group (4) were transported in farm water containing 10 ppm iodine. Blood samples were collected for the determination of serum cortisol and glucose, while skin mucus was collected for assaying lysozyme, peroxidase and antibacterial activity; cumulative mortality rate; and food reflexes which were assessed at 0 h, 1 h, 6 h, 48 h, 7 days and 14 days post 2 h transportation. The results indicated that fish tranquilization with MS-222 and/or treatment with iodine mitigated the stress condition associated with transportation and accelerate restoration of the normal physiological and immunological status. So, it was recommended to use the MS-222 as a tranquillizer together with iodine as an antiseptic during transportation of O. niloticus.