The current study aimed to assess the inhibitory effect of a DNA aptamer (Apt17) which targeted Salmonella invasion proteinA (SipA). The effect of Apt17, on biofilm formation by two Salmonella enteritidis strains, was tested either separately or in combination with ampicillin at different Sub MIC concentrations. Maximum inhibitory effect equivalent to 24.34% and 26.81% was recorded when Apt17 was co-incubated with S. enteritidis TM 6 and S. enteritidis TM 68 respectively for 13 h. The inhibitory effect of Apt17 was also confirmed with Triphenyl Tetrazolium Chloride. Under Scanning Electron Microscope, the presence of Apt17 resulted in altered three dimensional structure. While the treated cells of S. enteritidis TM 6 were arranged as monolayers, the sessile aggregates of S. enteritidis TM 68 appeared thinner and exhibited less surface coverage when compared to control. Moreover, the treated cells lost their exopolysaccharide matrix. The co-incubation of Apt17 with ampicillin MIC/10 for 24 h, inhibited the biofilms of S. enteritidis TM 6 and S. enteritidis TM 68 by 12.5 and 20.9% respectively. This study demonstrated quantitative and qualitative antibiofilm effect of Apt17 against the biofilms of two Salmonella enteritidis strains. According to our knowledge, this is the first study employing an aptamer that targets SipA protein to inhibit biofilm formation in Salmonella.