Purpose: This study was aimed at investigation of the effect of tetracycline (TTC) on myocardial infarct size (IS) in obese rats with chemically induced colitis (CIC). We also addressed the association between TTC-induced IS changes and plasma levels of cytokines and short-chain fatty acids (SCFAs), as well as intestinal microbiome composition. Methods: Obesity was produced in Wistar rats by 5-week feeding with high-fat, high-carbohydrate diet (DIO). Single rectal administration of 2 ml of 3% acetic acid resulted in groop CIC. Healthy (TTC) and comorbid rats received TTC (DIO+CIC+TTC) via gavage (15 mg daily for 3 days). The rats were euthanized followed by isolated heart perfusion with simulated global ischemia and reperfusion. Infarct size (IS) was determined histochemically. Lipopolysaccharide (LPS)/cytokine and SCFAs plasma levels were measured with ELISA and gas chromatography/mass spectrometry, respectively. Intestinal microbiome was analyzed using RT-PCR. Results: Administration of TTC to intact animals resulted in significant IS limitation (50 ± 7 vs. 62 ± 4% in controls, p < 0.05). IS was not different from controls in comorbid animals. TTC treatment in comorbid rats caused significantly larger infarct size in comparison to controls (77 ± 5%, p < 0.05). Elevated serum concentrations of proinflammatory cytokines and LPS were found in obese rats with CIC. Combination of obesity and CIC was characterized by lower abundance of Lactobacillus spp., Bifidobacterium spp. and increased counts of Escherichia coli vs. controls. The effect of TTC on IS was not associated with specific changes in SCFA levels. Conclusion: TTC treatment reduced IS in healthy rats. This effect was reversed in obese animals with CIC, which was associated with specific changes in microbiota and significantly elevated levels of cytokines and LPS.