Objective To investigate the diversity of gut microbiota and its association with the change in serum short-chain fatty acids in patients with acute myocardial infarction (AMI). Methods A total of 35 patients with AMI who were diagnosed and treated in Department of Cardiology, The People’s Hospital of Hongze District, from August 2023 to April 2025 were enrolled as AMI group, and 35 individuals who underwent physical examination during the same period of time were enrolled as control group. The two groups were compared in terms of the changes in the levels of gut microbiota (Bacteroides, Enterobacter, Enterococcus, Clostridiaceae, Bifidobacterium, and Lactobacillus) and short-chain fatty acids (acetic acid, butyric acid, propionic acid, valeric acid, and caproic acid), and the correlation between gut microbiota and short-chain fatty acids was analyzed. Results Compared with the control group, the AMI group had significantly lower numbers of Bacteroides (4.08±0.79 Ig CFU/g vs 7.36±1.25 Ig CFU/g, t = 13.160, P<0.05), Bifidobacterium (3.68±0.85 Ig CFU/g vs 12.41±1.63 Ig CFU/g, t = 28.157, P<0.05), and Lactobacillus (4.75±1.23 Ig CFU/g vs 14.98±1.96 Ig CFU/g, t = 26.158, P<0.05), significantly lower levels of acetic acid (4732.74±147.33 µg/mL vs 5387.96±261.74 µg/mL, t = 12.905, P<0.05), butyric acid (1675.97±182.27 µg/mL vs 1912.05±217.13 µg/mL, t = 4.927, P<0.05), propionic acid (427.59±98.42 µg/mL vs 501.96±143.99 µg/mL, t = 2.523, P<0.05), valeric acid (43.69±8.06 µg/mL vs 56.23±12.36 µg/mL, t = 5.027, P<0.05), and caproic acid (407.07±163.97 µg/mL vs 638.74±175.67 µg/mL, t = 5.704, P<0.05), and significantly higher numbers of Enterobacter (17.29±2.74 Ig CFU/g vs 5.29±1.63 Ig CFU/g, t = 22.266, P<0.05), Enterococcus (15.79±2.41 Ig CFU/g vs 4.79±0.98 Ig CFU/g, t = 24.991, P<0.05), and Clostridiaceae (8.10±1.63 Ig CFU/g vs 5.69±1.23 Ig CFU/g, t = 6.985, P<0.05). The Pearson correlation analysis showed that Bacteroides, Bifidobacterium, and Lactobacillus were positively correlated with acetic acid, butyric acid, propionic acid, valeric acid, and caproic acid (P <0.05), and Enterobacter, Enterococcus, and Clostridiaceae were negatively correlated with acetic acid, butyric acid, propionic acid, valeric acid, and caproic acid (P<0.05). Conclusion Gut microbiota dysbiosis is observed in patients with AMI, i.e., the reduction in probiotic bacteria (Bacteroides, Bifidobacterium, and Lactobacillus) and the proliferation of harmful bacteria (Enterobacter, Enterococcus, and Clostridiaceae), and the serum levels of short-chain fatty acids (acetic acid, butyric acid, propionic acid, valeric acid, and caproic acid) are affected by gut microbiota dysbiosis.