Pumpkin seed oil has long been considered as an ingredient for its nutritional and medicinal values for the prevention of various ailments, especially for prostate diseases. In addition, several studies have suggested the crucial roles and effectiveness of pumpkin seed oil in the treatment of diabetes, anxiety and even cancer. Pumpkin seed oil is being used in several countries worldwide including North America, Mexico, India and China. This review highlights the characterization, properties and use of pumpkin seed oil from various pumpkin species against several diseases pathophysiologies. We strongly believe that this review will provide overall insights to the chemists, biologists and researchers on the roles of pumpkin seed oil extracts that possess promising biological activities.