Most species of Gram-negative bacteria produce the enzyme β-lactamase, which can inactivate the structure of β-lactam antibiotics. Pseudomonas aeruginosa has revealed countless resistances to β-lactam antibiotics and others over the last few decades. Therefore, the present study aimed to determine the prevalence of ampicillin β-lactamase (AmpC) enzymes, conduct a molecular investigation of the AmpC gene expressing the enzymes and assess their impact on the widespread occurrence of antibiotic resistance in P. aeruginosa. One hundred and fourteen P. aeruginosa isolates were collected from wounds, UTIs, burns, ears, and sputum and identified using various culture media, tests, and the VITEK 2 compact system. The isolates were identified, exhibiting high resistance rates of 100% to Ampicillin and 95.4% to Nalidixic acid. They also demonstrated resistance rates of 18.2% to Chloramphenicol and 22.7% to Ciprofloxacin. The Iodometric method test revealed that 63.1% produced β-lactamase enzymes, while the remaining 36.8% did not produce β-lactamase enzymes. The finding of the CHROM agar and Extended-Spectrum β-Lactamases) ESBLs (tests showed that 100% of isolates produced broad-spectrum β-lactamase. The double disk synergy test (DDST) for detecting AmpC enzymes showed that 19.2% tested positive, while 80.7% were unable to produce these enzymes. Molecular detection of the AmpC gene revealed that 81.8% of the isolates possessed this gene, while 18.1% did not. Out of the 22 isolates of P. aeruginosa studied, 18 exhibited a high percentage of the AmpC gene. This gene produces penicillinase enzymes, contributing to resistance against most studied antibiotics.