This study was carried out to detect the presence of aflatoxin B₁ (AFB₁) in 36 samples of spices from Iran and India that include chilli powder (n = 12), black pepper powder (n = 12) and whole black pepper (n = 12). Enzyme-linked immunosorbent assay was applied to analyse AFB₁ in the samples. All the analyses were done twice. AFB₁ was found in all the spices samples, the concentration of AFB₁ in Iranian samples was ranged from 63.16 to 626.81 ng/kg and in Indian samples was ranged from 31.15 to 245.94 ng/kg. The mean of AFB₁ concentration in the chilli powder was significantly higher (p < 0.05) than the whole and powdered black pepper. However, none of the samples exceeded the maximum prescribed limit, that is 5000 ng/kg (5 µg/kg) of European Union regulations for AFB₁. Although, the present research was not a comprehensive study; however, it provides valuable information on AFB₁ levels in Iranian and Indian spices.