The flavor and quality of the Iranian enzymatic cheese are influenced by different factors during its production and storage. The present study was aimed to investigate the effects of various processing conditions, such as pasteurization, retentate treatment, microfiltration (MF), double bactofugation (DBF) and ultrafiltration on the microbial, chemical and textural analysis of the Iranian enzymatic cheese. Our results revealed a significant reduction of the mesophilic bacterial counts (p < 0.05), in all the applied methods of processing. A considerable increase in the numbers of yeasts and molds was observed up to the 21st days of storage (p < 0.05). No obvious changes were observed in the spore counts in 4 °C storage condition, except for the treatment that double bactofugation was excluded. Moreover, the levels of proteins and NPN remained stable. Level of lactose was reduced during the shelfing which was associated with the increased acidity due to the lactose fermentation. In general, the rigidity of samples was gradually increased up to the end of storage (p < 0.05). The current study revealed that concomitant use of different technologies (Hardles’ method) improved the overall acceptance of the cheese and increased its shelf life, the qualities which enhances at 4 °C storage condition.