AbstractWaste frying oil (WFO) is a degraded form of oil that is produced from repeated exposure to high temperatures during cooking. Many research studies have focused on the use of recycled WFO. These studies are highly promoted because of the need to find new ways of reducing the negative impact of WFO on the environment. One of these studies focused on the production of sophorolipid (SL), a naturally derived eco‐friendly glycolipid biosurfactant. Unfortunately, the environmental and societal advantages brought about by the wide‐spread use of SL are offset by its high production cost. WFO is a high‐volume, inexpensive material that can be used as a substrate for SL production. As such, by utilizing WFO as a feedstock material cost reduction can be realized to improve large‐scale application potential. One drawback to the use of WFO is that its physical characteristics are different from fresh oil and its effect on SL productivity (g/day) has not been investigated. This research focused on the effect of acid value (AV), peroxide value (PV), and carbonyl value (CV) of WFO on SL productivity. It was confirmed that SL titers (111.1 vs. 106.7 g/L) did not differ significantly after using either fresh oil (CV <0.01) or WFO, respectively. In addition, this research also confirmed that higher WFO degradation required longer culture periods (from 6 days to 9 days) to reach comparable SL concentrations as that produced with fresh oil.