Background Biomaterial-associated infections are one of the most important complications in orthopedic surgery. The main goal of this study was to demonstrate the in vivo bactericidal effect of ultraviolet (UV) irradiation on Ti6Al4V surfaces. Materials and methods An experimental model of device-related infections was developed by direct inoculation of Staphylococcus aureus into the canal of both femurs of 34 rats. A UV-irradiated Ti6Al4V pin was press-fit into the canal by retrograde insertion in one femur and the control pin was inserted into the contralateral femur. To assess the efficacy of UV radiation, the mean colony counts after inoculation in the experimental subjects and the control group were compared at different times of sacrifice and at different inoculum doses. Results At 72 h, the mean colony counts after inoculation in experimental femurs were significantly lower than those of the control group, with a reduction percentage of 76 % (p = 0.041). A similar difference between control and experimental pins was observed at 24 h using an inoculum dose <10⁴ colony-forming units (CFU), for which the reduction percentage was 70.48 % (p = 0.017). Conclusion The irradiated surface of Ti6Al4V is able to reduce early bacterial colonization of Ti6AlV pins located in the medullar channel and in the surrounding femur. The reductions depend on the initial inoculums used to cause infection in the animals and the greatest effects are detected for inoculums <10⁴ CFU. Level of evidence Not applicable.