Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The most important risk factor for the development of HCC is cirrhosis regardless of etiology. 5-Fluorouracil (5-FU) is widely used in the treatment of cancer. Drug resistance remains a significant limitation to the clinical use of 5-FU. The present study aimed to evaluate the therapeutic efficacy of 5-FU loaded chitosan nanoparticles in experimentally induced HCC. To achieve our purpose, one hundred and five male Swiss Albino mice were divided randomly into two major groups:  Group A: comprised 25 mice served as normal control, Group B: comprised 80 mice received a daily oral dose of 0.06% DAB (165 mg/kg body.wt.) for 30 days after which the water was replaced with 0.05% aqueous solution of Phenobarbital (PB). Five chosen mice randomly from groups A and B at the time intervals 15, 30, 45 and 60 days were sacrificed to follow up with the development of HCC by biochemical and histopathological examination. Animals of group B were divided into 3 groups Group I: included 20 mice served as an untreated group, group II: included 20 mice injected intraperitoneally with 5-FU only (40mg/kg body.wt) every 2 days for 16 days, group III:  included 20 mice injected intraperitoneally with 5-FU Cs NPs. Each group was further divided into two subgroups 10 mice each, one subgroup treated with ultrasonic waves; meanwhile the other subgroup without ultrasonic waves exposure. At the end of the experiment, animals were sacrified, serum ALT, hepatic ALT, and hepatic MDA were estimated; HCC was histopathologically monitored in all studied groups. There was 276.5%, 145.7%and 438.5% increase in serum ALT, hepatic ALT and hepatic MDA levels respectively comparing to the corresponding control. Liver tumors that ultimately became neoplastic were produced after 45 days. US exposure triggered a significant decline in serum and hepatic ALT activity (P = 0.001) and in hepatic MDA (P = 0.009) within 5-FU loaded Cs NPs group. Moreover, tumor growth delay and more enhanced correction in hepatic architecture was obtained by a combination of US and 5-FU loaded Cs NPs therapy. Based on these results, we can conclude that the use of 5-FU loaded chitosan nanoparticles in combination with low-intensity ultrasound ameliorates the efficacy of 5-FU as anticancer therapy for HCC.