This study was conducted at the poultry farm located in the College of Agricultural Engineering Sciences, University of Baghdad, Abu Gharib (the old site), and laboratories of the Animal Production Department, Jadriya, to investigate the effect of adding hydrogen peroxide H2O2 at nanoscale levels to semen diluents of local roosters sperm in a number of semen characteristics. In this study, 80 roosters local Iraqi chickens were used, the roosters were trained three times a week, to collect semen, until the largest number of them responded. Then the best 40 of the roosters were elected for the purpose of collecting the semen with a pooled sample, and then the samples were diluted and divided equally into four parts. The concentrations of 0, 1, 10, 100, nM of H2O2 were added to each part of the diluted semen, then kept cool until the temperature reached 5 C for three periods (0, 24, 48 hours), and cryopreservation (48 hours) for all four addition levels. A number of laboratory characteristics were studied including percentages of individual motility, dead sperm, mitochondrial efficacy, and DNA Fragmentation at the end of each repetition (10 repetitions). A variation based on the concentration was observed in the results of hydrogen peroxide, as it ranged from the non-affectivity of the two treatments 1, 10 nM H2O2, to the deterioration in some laboratory characteristics for the treatment of 100 nM H2O2, and according to the interactions between the addition concentrations and the cooling and cryopreservation periods. From this experiment, it can be concluded the inefficiency of the hydrogen peroxide concentrations used to semen preservation.