Appendix is a small, closed tube that is about the size of 9 cm. It attaches to the beginning of our large intestine, where the small and large intestines meet. Appendicitis, an inflammation of appendix is mostly related with bacterial etiological agents. The present study is aimed to study bacterial etiological agent that infected the vermiform appendix. Sterile Swabs were used to collect samples from various abdominal sites in addition to the lump of infected appendix. The clinical samples were collected from 69 patients diagnosed as suspected appendicitis from various hospitals in Surat region. Bacterial etiological agents were isolated and identified by standard microbiological procedures. Aamong 69 samples collected, 62 isolates were identified with predominance of gram negative bacteria (n=39), however Staphylococcus aureus found as prime pathogen among all. Most of the gram negative isolates found more resistant to Ofloxacin while gram positives against Gentamycin. 47.8% of MDR and 51% of ESBLs producers were knocking outcomes of our study. Further the detection of biofilm formation capabilities of these isolates were carried out by Tube method and Higher degree of biofilm production observed by tube method in compare to microtiter plate method.