In this study the influence of different concentrations of three gas mixture (carbon dioxide, nitrogen,oxygen) ,and also vacuum conditions and flexible multi-layer films on growth of anaerobic bacteria of smoked kutum fish (Rutilus frisii kutum) at ambient condition (T= 25 0C) were evaluated. Ordinary condition as control packaging were compared with four kinds of modified atmosphere packaging: (N270%+ CO230%), (N230% + CO270%), (45%CO2+45%N2+10%O2),and vacuum conditions, in this study. These samples(smoked kutum fish) were packaged in 3 types of flexible multi-layer films under modified atmosphere packaging,3- layers(PET(12)/AL(12)/LLD(100))and 4-layers (PET(12)/AL(7)/PET(12)/LLD(100) )and 3- layer( PET(12)/AL(7)/LLD(100)). Packed samples were performed microbial tests (Anaerobic bacteria count), in different times during 60 days, with 15 treatment,3 run, statistical analysis and comparison of data, were done by software SAS (Ver:9/1) and Duncan’s new multiple range test, with confidence level of 95% (P <0.05).The usage of MAP was not adequet for controlling spolage but the spoilage process was delayed. The shelf life of smoked kutum fish(according to anaerobic bacteria count) in 4-layers , under conditions 1 , 2 & 3 were reported 60,58,45 days and in vacuum conditions about were 40 days, in 3-layers (AL:12) , under conditions 1,2 &3 and in vacuum conditions were 55,50,40,35 days, with 3- layers(AL:7),under conditions 1,2 &3 45,40,35 days and in vacuum conditions were 30 days. Anaerobic bacteria count showed that increasing CO2 concentration increased shelf life. According to these results could be concluded the best condition for anaerobic bacteria count belonged to treatment under modified atmosphere CO2 70% and 4- layers due to thickness (131 μ), low steam permeability in this 4-layer container and antibacterial properties of more amount of carbon dioxide.