© 2021 Wiley Periodicals LLC.In this study, the effect of cholesterol on antibiotic resistance of Salmonella Typhimurium was investigated. Antibiotic resistance patterns revealed that the bacterium was susceptible to ampicillin, streptomycin, sulfamethoxazole/trimethoprim, ciprofloxacin, tetracycline, chloramphenicol, and gentamicin and resistant to penicillin, erythromycin, and vancomycin. The minimum inhibitory concentrations (MICs) of ampicillin, streptomycin, ciprofloxacin, and sulfamethoxazole/ trimethoprim antibiotics were found as 16, 128, 0.25, and 16 mg/L, respectively. When the effect of cholesterol on antibiotic resistivity was examined, it was found that MICs increased in cholesterol medium than in non-cholesterol medium. The increase in MICs and decrement in inhibition zones was considered as the decreasing effect of cholesterol on the sensitivity of Salmonella typhimurium. The effect of cholesterol on the antibiotic sensitivity of Salmonella typhimurium was also assessed by Fourier transformed infrared (FT-IR) spectroscopy, and the results indicated that new functional groups were observed at different wavelengths in the presence of cholesterol. Practical applications: Antibiotic resistance is a global phenomenon resulting in the emergence of pathogens with resistance to clinically important antibiotics. Antibiotic resistance in foodborne pathogens such as Salmonella is a major concern for public health safety. Significant increase in bacterial resistance makes it increasingly necessary to develop new drugs and antibiotic activity modifiers and effective techniques for treatment. Fourier transformed infrared (FT-IR) can be used as a rapid and precise method for the assessment of cholesterol effect on antibiotic resistance pattern of Salmonella typhimurium.