<p>Given that chickens can easily become a culture medium for microbial growth and reproduction, the prevention and control of biological safety risks in chickens is important. Chlorine-containing antimicrobial agents have been banned in some countries due to safety risks, while the efficacy of non-chlorine alternatives in the mixed precooling process (water cooling followed by air cooling) of chicken slaughter remains understudied. This study aimed to investigate the effect of adding sodium hypochlorite or non-chlorine antimicrobial agent (acetic acid, lactic acid or peracetic acid) to water cooling in a three-stage spiral pool, followed by air cooling, on the microbial diversity in yellow-feathered chickens. Results showed that sodium hypochlorite had the best antibacterial effect, evidenced by 3.50 log CFU/cm² of total plate count and 2.62 log CFU/cm² of coliform groups after treatment. Peracetic acid had the best antibacterial effect on <i>Pseudomonas</i>, reducing the number of colonies to 2.72 log CFU/cm² after treatment. It also had the best comprehensive effect on the sensory quality of chicken breast meat with a total score of 33.4. High-throughput sequencing was applied to compare the antibacterial effects of these antimicrobial agents. The microbial community richness under the action of sodium hypochlorite and Acetic acid ranked in the top two among the four treatments. <i>Proteobacteria</i>,<i> Bacteroidetes</i>,<i> Actinobacteria</i> and <i>Firmicutes</i> were mainly distributed in the carcass surface of the yellow-feathered chickens treated with the antimicrobial agents. This study provided valuable information for understanding the microbial composition of chicken carcasses under mixed precooling conditions and selecting effective antibacterial agents to ensure microbial safety.</p>