Roads affect the population of wild animals and are source of barrier between animal's leads to reduction of biodiversity results in population isolation. To observe roadside accidental biodiversity lossand estimate the total number of accidental biodiversity lossbyproviding solution to reduce the roadside biodiversity loss. A study was designed to measure the roadside accidental biodiversity loss from Bahawalpur to Lal Sohanra road with a length of 30 Kms and Yazman to Bahawalpur with a length of 31km distance) where two way traffic mode which kept under observation for the collection of data. Average mortality ratio was calculated and it was observed that Reptiles, Birds and Mammals have 2.88±1.918, 3.71±2.956 and 7.46±3.878 mortality per month respectively on both studied transects (Bahawalpur to Lal Sohanra and Yazman to Bahawalpur). Road has a profound effect on the mortality fauna and wildlife loss occur due to neglegance of Drivers. Foraging behavior of Animals one of the major cause to these exposure of roads. Propre counseling of driver in relation to importance of fauna, vistors should avoid putting partially consumed food bags on road, solid waste management should adequqte, and fancing of roads by trees to avoid exposure of fauna to roads.