Multidrug-resistant (MDR) bacteria have been isolated from a major wastewater pathway. These bacteria harbor several antimicrobial resistance genes that confer resistance to several antibiotics simultaneously. The main aim of this study was to investigate the antibiotic resistance profiles of lactose-fermenting gram-negative bacteria isolated from the main wastewater pathway in the Nablus area of the West Bank, Palestine. A total of 162 lactose-fermenting isolates belonging to the Enterobacteriaceae family were isolated from a sample obtained from the main wastewater pathway. Most of the isolates obtained were identified to the species level using the API-20 E identification system. The proportions of MDR strains among the obtained Escherichia coli and Citrobacter Koseri isolates were 19.1% and 10%, respectively. Among all isolates, six were found to be extended-spectrum beta-lactamase (ESBL) producers. These included three E. coli isolates, one Klebsiella pneumoniae isolate, and two C. Koseri isolates. Approximately 12.3% of the total isolates were MDR and 3.7% were identified as ESBL producers. The prevalence of MDR isolates in our study was concerning, indicating that immediate and decisive measures are needed to halt its escalation and promote its reduction.