This study aimed to determine phosphorus and vitamin B12 supplementation effect in semen extender on the quality and fertility ability of chilled Thai native rooster semen. Eighty-four ejaculates of semen from 26 Thai native roosters (Burmese x Vietnam crossbreed) were included. Semen was collected by applying dorsal–abdominal massage once a week, pooled, diluted to 500 million sperms per dose, and divided into six groups. The semen samples used for control group were diluted with modified Beltsville poultry semen extender (BPSE). For the treatment groups 2–6: semen samples were diluted with modified BPSE and enriched with phosphorus and vitamin B12 (Octafos® Octa Memorial Co., Ltd., Bangkok, Thailand) at concentrations 0.02%, 0.04%, 0.06%, 0.08%, and 0.10%. Semen fertility ability was tested in six replications by inseminating layer hens. Thirty-six Thai native hens were randomly assigned to three groups (control, 0.04%, and 0.08%) of 12 hens and were inseminated with a dose of 0.2 mL on collecting day. Sperm motion characteristics (i.e., sperm motility, sperm progressive motility, and sperm kinetic parameters) were measured using a computer-assisted sperm analysis system (SCA®, Proiser S.L., Valencia, Spain). Sperm viability, mitochondrial activity, acrosome integrity, plasma membrane integrity, and malondialdehyde (MDA) concentration were also evaluated. The sperm motion characteristics were the highest in the 0.04% supplementation group on all days of collection, especially the VCL and VAP (P < 0.05). The viability, mitochondrial activity, plasma membrane and acrosome integrity of spermatozoa were greater in the 0.04% supplementation group than in the control groups (P < 0.05). The 0.04% supplementation group had the lowest MDA concentration in all days of collection. The 0.04% supplementation group were higher both fertility (66.59% vs. 48.50%: P < 0.05) and hatching rates (58.80% vs. 43.18%: P < 0.05) than in the control group. In conclusion, 0.04% phosphorus and vitamin B12 concentrations supplementation in semen extender improved rooster semen quality and fertility in chilled rooster semen.