Pancreatic cancer remains one of the most lethal forms of cancer with a 10-year survival of <1%. With little improvement in survival rates observed in the past 40 years, there is a significant need for new treatments or more effective strategies to deliver existing treatments. The antimetabolite gemcitabine (Gem) is the most widely used form of chemotherapy for pancreatic cancer treatment, but is known to produce significant side effects when administered systemically. We have previously demonstrated the benefit of combined chemo-sonodynamic therapy (SDT), delivered using oxygen carrying microbubbles (O2MB), as a targeted treatment for pancreatic cancer in a murine model of the disease. In this manuscript, we report the preparation of a biotin functionalised Gem ligand for attachment to O2MBs (O2MB-Gem). We demonstrate the effectiveness of chemo-sonodynamic therapy following ultrasound-targeted-microbubble-destruction (UTMD) of the O2MB-Gem and a Rose Bengal loaded O2MB (O2MB-RB) as a targeted treatment for pancreatic cancer. Specifically, UTMD using the O2MB-Gem and O2MB-RB conjugates reduced the viability of MIA PaCa-2, PANC-1, BxPC3 and T110299 pancreatic cancer cells by >60% (p < 0.001) and provided significant tumour growth delay (>80%, p < 0.001) compared to untreated animals when human xenograft MIA PaCa-2 tumours were treated in SCID mice. The toxicity of the O2MB-Gem conjugate was also determined in healthy non-tumour bearing MF1 mice and revealed no evidence of renal or hepatic damage. Therefore, the results presented in this manuscript suggest that chemo-sonodynamic therapy using the O2MB-Gem and O2MB-RB conjugates, is potentially an effective targeted and safe treatment modality for pancreatic cancer.