Background: Lime juice is a nutritious drink, which is generally consumed for its' refreshing properties, nutritive value, vitamin content, and health benefits. Therefore, the chemical and microbiological quality of the traditional and industrial lime juice produced in Kashan city was assessed. Methods: In this descriptive cross-sectional study, a total of 106 samples were collected and screened for total soluble solid (TSS), pH, acidic value, protein content, mold, yeast, and count of acid-tolerant bacteria according to the Institute of Standards and Industrial Research of Iran (ISIRI). Results: Of the total samples, 66 samples (62.26%) were within the Iran’s national standard range. Most samples that did not meet the national standard requirements, were related to traditional samples (70.24%) compared to industrial samples (31.82%, P < 0.001). No significant difference was found between pH and protein of traditional and industrial lime juice samples (P > 0.05), while TSS and acidic values in traditional lime juices were less than those of the industrial lime juice (P < 0.05). Additionally, the mold and yeast contaminations in traditional lime juices were more than industrial lime juices (P < 0.05). Conclusion: In overall, traditional lime juice samples had the most microbial and chemical contaminations. It is necessary to increase the regular monitoring by relevant organizations over quality of the produced lime juices.