Pseudomonas aeruginosa is a common pathogen of a wide range of nosocomial infections in humans, as well as foodborne illnesses. The current study focuses on the molecular genotyping of P. aeruginosa recovered from human, water, and food specimens. A total of 350 samples, fifty samples each from human, tap water, fish swamp, chicken meat, minced meat, raw milk, and hospital surface from the Menoufiya governorate, Egypt. P. aeruginosa was detected in 14.28%, including human (26%), tap water (18%), fish swamp (18%), chicken meat (12%), minced meat (10%), raw milk (16%), and hospital surface (0%). The results of testing of 50 P. aeruginosa isolates against sixteen antibiotics revealed a relatively high antibiotic resistance for Amoxicillin (100%), Erythromycin (98%), Cephradine (90%), Colistin (82%), Oxytetracyclin, (79%), Chloramphenicol (70%), Doxycycline (70%), and Kanamycin (62%) and high susceptibility for Imipenem (96%), Apramycin (94%), Amikacin (90%), Norfloxacin (78%), Sulphamethoxazol (86%), Enrofloxacin (64%), and Ofloxacin (60%). Furthermore, PCR was successfully amplified for the toxA, exoS, and oprL virulence genes at 396, 118, and 504 bp, respectively, as well as amplifying the ermB, pelA, blaTEM, and tetA resistance genes at 639, 786, 516, and 570 bp, respectively. The dendrogram investigation by ERIC-PCR of 10 clinical P. aeruginosa isolates revealed two main clusters and 10 different ERIC-PCR patterns. The presence of P. aeruginosa isolates in food may represent a potential public health concern, with the need for further epidemiological studies, as well as whole-genome sequencing and correlations of P. aeruginosa in water, food samples, and human infections.