Nonsurgical fertility control is increasingly advocated as more cost-effective than surgical sterilization to manage stray animal populations in a different part of the world. An experimental study was conducted from December 2018 to April 2019 at Mekelle University to evaluate the effect of single bilateral intratesticular injection of cetrimide 2% in adult albino mice. A total of 20 clinically healthy albino mice selected based on their age and sex and were divided randomly into five groups and evaluation was conducted for 30 days after intratesticular injection of cetrimide solution 2% at the dose rate of 5, 10, 15 and 20 mg per testis and for control 0.1 mL normal saline per testis per 100 g body weight were given. All albino mice were evaluated for 30 days at a fixed interval. Change in body weight, scrotal width, sexual behavior, and fertility performance was also assessed. On day 30, all albino mice were sacrificed for histopathological study. Means  ±  Standard deviation of the mean, one-way, and a mixed model ANOVA (for repeated measures) was used to summarize the data, determine the effects of group and time on bodyweight and scrotal width. The significant increase in body weight (p - 0.001) and significant reduction of scrotal width (p - 0.001) were noted in all cetrimide treated in comparison to control groups. In addition, there was a significant (p < 0.05) reduction of scrotal width in albino mice after intratesticular injection of cetrimide on day 1, 10, 15, 20, 25, and 30 with respect to their experimental groups. Testicular histology revealed that there were multinucleated giant cells in seminiferous tubules, derangement of tubular architecture along with infiltration of leucocytes and appearance of fibrous tissue were seen on testicular sections at a dose rate of 15 and 20 mg. Similarly, a significant change in the sexual behavior of the treated males and no pregnancy was detected on female albino mice after 21 days post-coital at 10, 15, or 20 mg cetrimide-treated males. In conclusion, a single bilateral intratesticular injection of cetrimide 2% at a dose of 15 and 20 mg might provide an effective way of sterilization and may be considered as an alternative to surgical castration in male animals. Besides, further assessment should be done in the future to identify the mechanism of infertility.