Biofilm production by Gram-positive Cocci in clinical samples is widespread around the world. It is a potential threat to human health. The objective of this study was to assess the antibiotic resistance of Gram-positive Cocci in clinical samples collected from Damietta governorate both prior to and following the production of biofilm, while also examining the genetic alterations of the ClpC gene. A total of 117 clinical samples were gathered, out of which 100 were found to have pathogenic bacterial strains (i.e., urine, throat swap, vagina swap) from male and female. Fifty strains were identified as Gram-positive cocci (Staphylococcus aureus, Staphylococcus epidermidis, Strreptococcus pneumoniae, Enterococcus faecalis, and Micrococcus varians). All strains examined demonstrated the capacity to generate biofilm. The strains underwent sensitivity testing against a total of nineteen commercially available antibiotics before and after bacterial biofilm formation. Meropenem (MEM-10 mg) was the most commonly used drug for the treatment of isolated pathogens and affected all strains, with 98% before biofilm formation and 92% after biofilm formation, while Cefazolin (CZ-30 mg) had the lowest effects (16% before biofilm formation and 6% after biofilm formation). The Multiple Antibiotic Resistance Index (MARI) was calculated for the strains under examination before and after biofilm formation. Furthermore, a quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to analyze the relative gene expression of ClpC in two samples of Staphylococcus aureus. ClpC is a crucial factor for stress tolerance, growth recovery, and cell death. The ClpC gene has an effect on bacterial biofilm.