Background & Aims of the Study: Wheat flour makes the basic component of bread. Thus, evaluating its quality plays an important role in public health. This paper focused on the physicochemical and microbial analysis of all bread flour types, used in the bakeries of Hamedan, Iran during 2015-2016. Materials & Methods: All bread flour types such as Barbari, Lavash, Sangak and Tafton bread flours were collected from 100 bakeries which baked various types of wheat bread in Hamedan, western Iran from May 2015 to February 2016 in this cross-sectional study. Moisture, dry matter, ash, fat and protein percent values of samples were analyzed. Total viable counts of bacteria as well as yeasts and molds of all samples were determined. Identification and characterization of enumerated fungi was also identified. Results: The highest moisture content percent belonged to Sangak flour (12.75±0.06), significantly (P<0.05) followed by Taftoon (12.57±0.03), Barbari (11.77±0.03) and Lavash (11.57±0.03), respectively. The order of ash percent was as follows: Lavash (1.36±0.01), Sangak (1.11±0.04), Barbari (1.03±0.03) and Taftoon (0.93±0.02). According to the results, Taftoon flour meaningfully (P<0.05) had the highest crude protein value (5.31±0.17%), followed by Sangak (4.11±0.03%), Barbari (2.75±0.05%) and Lavash (2.40±0.09%), respectively. Protein and fat deficiency were observed in all flours. Total viable counts of bacteria were in permissible range in all flours. Yeasts and molds of all samples exceeded recommended limits. Conclusion: It was found that the fungal growth control is essential by improving storage conditions, hygienic operations and nutrients enrichment in wheat flours of all bread types of Hamedan, Iran.