Urinary tract infection (UTI) is one of the most common bacterial infections in the world, which is associated with high morbidity and mortality rates. Enterobacterales species are considered the most causative agent for UTI, especially uropathogenic Escherichia coli (UPEC). Here, we investigated the antibacterial activity of the green fungal metabolite, 6-pentyl α pyrone lactone, alone or in combination with zinc oxide nanoparticles (ZnONPs) against multidrug-resistant Enterobacterales recovered from UTI. The results revealed that 57.27% of human urine samples were positive for Enterobacterales, where E. coli was the most prevalent bacterial pathogen (66.67%). Of note, 98.41% of Enterobacterales isolates were multidrug-resistant (MDR) with multiple antimicrobial resistance (MAR) indices ranged from 0.437 to 1. Fifty percent of the examined isolates were positive for the integrase gene; 60% out of them harbored class 2 integron, whereas the other 40% carried class 1 integrons. The broth microdilution assay ensured that the 6-pentyl-α-pyrone lactone had a reasonable antimicrobial effect against the examined isolates (Minimum inhibitory concentration (MIC) values of 16–32 μg/mL). However, ZnONPs showed a strong antimicrobial effect against the investigated isolates with MIC values ranging from 0.015 to 32 μg/mL. Interestingly, the MICs decreased 5–12 fold and 3–11 fold for 6-pentyl-α-pyrone lactone and ZnONPs, respectively, against examined isolates after their combination. This is the first report suggesting the use of 6-pentyl α pyrone lactone and ZnONPs combination as a promising candidate against MDR Enterobacterales recovered from UTI.