Tomato seeds, which are the byproducts of tomato paste processing, were used to obtain tomato seed oil using two different extraction methods: cold press extraction (CPE) and enzyme-assisted aqueous extraction (EAAE). To maximize oil amount, the experimental parameters of those two methods were optimized separately by response surface methodology. The optimized methods were used to evaluate their quality characteristics. The maximum oil amounts for tomato seed oil using CPE and EAAE were found as 12.80% and 9.66%, respectively. These obtained results present important information for the determination quality characteristics including the physical, chemical, microbiological, and bioactivity properties of tomato seed oil in both extraction methods used. The comparison of two methods shows that they are both ecology friendly extraction methods. It also helps to a great extend to evaluate quality characteristics and bioactivity properties of tomato seed oil. Therefore, tomato seed oils extracted by two methods are promising for future usage as functional food. This manuscript will comprise the basis of our future studies analyzing the new compounds obtained from agro-food waste.