P. aeruginosa is a famous bacterium that causes several diseases and has a high ability to be a multidrug resistant organism that is linked with the formation of biofilm. This study aimed to investigate tssC1 gene role in the resistance of different antibiotics in the presence of biofilm. We constructed biofilm for the isolates under the study and showed the effect of different antibiotics on biofilm formation and maturation. The presence of the gene was detected through achieving PCR reaction. Finally, tssC1 gene variation was determined through sequencing and aligning the sequencing products. The results showed that most of the isolates (80%) formed biofilm that played a role in the resistance of different antibiotics which could be due to the presence of tssC1 gene. However, the genic variation of tssC1 gene showed that no variation was detected. Therefore, we think this gene has no a role in the resistance of antibiotics and that the resistance may have been raised by other mechanisms found in P. aeruginosa isolates. This led us to conclude that the tssC1 gene does not contribute to the resistance of antibiotics through biofilm.