At present, the low post-thaw quality of poultry semen presents a challenge to develop new strategies for its cryopreservation. The purpose of this research was to assess the impact of kaempferol (KAE) on post-thaw turkey sperm characteristics (motility, membrane and acrosome integrity, mitochondrial function), oxidative and microbial profile. Turkey semen (n = 40) was diluted and cryopreserved in modified Beltsville extender with 5, 10, and 25 µM of KAE or without it (cryopreserved control – CtrlC), while fresh semen served as negative control (CtrlN). Following thawing, parameters were evaluated including sperm motility, membrane and acrosome integrity, mitochondrial functionality, DNA fragmentation index, apoptosis status, global reactive oxygen species (ROS) generation, lipid peroxidation (LPO) and protein oxidation. Bacterial identification was performed by matrix-assisted laser desorption/ionisation mass spectrometry. Our data suggest that motility, membrane and acrosome integrity, mitochondrial activity continuously increased correspondingly to KAE concentration versus CtrlC (P < 0.05) while cell apoptosis, ROS generation, LPO and protein oxidation were significantly decreased in KAE treated groups versus CtrlC (P < 0.05). Bacterial growth was suppressed in all KAE-treated groups, which acted synergistically with penicillin to eradicate most bacterial strains from cryopreserved samples versus CtrlN. Finally, our results suggest that KAE possesses strong antioxidant and antimicrobial properties which may be used to improve commercially available extenders for more effective preservation of turkey spermatozoa.